Cancer Treatment Methods Using Thermotherapy And/Or Enhanced Immunotherapy

ABSTRACT

A method of therapy for a tumor or other pathology by administering thermotherapy or a combination of thermotherapy and immunotherapy optionally combined with gene delivery. The combination therapy beneficially treats the tumor and prevents tumor recurrence, either locally or at a different site, by boosting the patient&#39;s immune response both at the time of original therapy and/or for later therapy. The therapy may further include the administration of a vaccine.

This patent application claims priority to U.S. Provisional PatentApplication No. 63/004,256, entitled “Cancer Treatment Methods UsingThermotherapy And Drug Delivery”, filed on Apr. 2, 2020, and is acontinuation-in-part of U.S. patent application Ser. No. 16/004,401,entitled “Early Cancer Detection And Enhanced Immmunotherapy”, filedJun. 10, 2018, which claims priority to U.S. Provisional PatentApplication No. 62/614,456, entitled “Cancer Treatment Methods UsingThermotherapy and/or Enhanced Immunotherapy”, filed on Jan. 7, 2018, andSer. No. 16/004,401 is a continuation-in-part of application Ser. No.15/853,821, entitled “Early Cancer Detection And EnhancedImmunotherapy”, filed Dec. 24, 2017, now U.S. Pat. No. 10,300,121, whichclaims priority to U.S. Provisional Patent Application No. 62/569,592,entitled “Cancer Treatment Methods Using Thermotherapy and/or EnhancedImmunotherapy”, filed on Oct. 8, 2017, and to U.S. Provisional PatentApplication No. 62/577,485, entitled “Cancer Treatment Methods UsingThermotherapy and/or Enhanced Immunotherapy”, filed on Oct. 26, 2017,and Ser. No. 15/853,821 is a continuation-in-part of application Ser.No. 15/143,981, entitled “Early Cancer Detection And EnhancedImmunotherapy”, filed May 2, 2016, now U.S. Pat. No. 9,849,092, which isa continuation-in-part of application Ser. No. 14/976,321, entitled“Method to Visualize Very Early Stage Neoplasm or Other Lesions”, filedDecember 21, 2015, now U.S. Pat. No. 10,136,820, the disclosure of eachof which is hereby incorporated by reference as if set forth in theirentirety herein.

This patent application also is a continuation-in-part of InternationalPatent Application Ser. No. PCT/US2018/054880, entitled “CancerTreatment Methods Using Thermotherapy and/or Enhanced Immunotherapy”,filed Oct. 8, 2018, which claims priority to U.S. Provisional PatentApplication No. 62/569,592, entitled “Cancer Treatment Methods UsingThermotherapy and/or Enhanced Immunotherapy”, filed on Oct. 8, 2017,U.S. Provisional Application No. 62/577,485, entitled “Cancer TreatmentMethods Using Thermotherapy and/or Enhanced Immunotherapy”, filed onOct. 26, 2017, U.S. Provisional Application No. 62/614,456, entitled“Cancer Treatment Methods Using Thermotherapy and/or EnhancedImmunotherapy”, filed on Jan. 7, 2018, and U.S. Provisional PatentApplication No. 62/720,258, entitled “Cancer Treatment Methods UsingThermotherapy and/or Enhanced Immunotherapy”, filed on Aug. 21, 2018;the disclosure of each of which is hereby incorporated by reference asif set forth in their entirety herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagrammatic illustration of a human body showing thetreatment of tumors located in various areas of the body with lightemitted by fiber optic devices, according to an embodiment of theinvention;

FIG. 2 is a diagrammatic illustration of a human eye showing thetreatment of tumors located in several areas of the eye with lightemitted by fiber optic devices, according to another embodiment of theinvention;

FIG. 3 is a diagrammatic illustration of several digestive organs of thehuman body showing the treatment of a tumor located in the pancreas withlight emitted by a fiber optic device, according to yet anotherembodiment of the invention;

FIG. 4 is a diagrammatic illustration of the urinary system of the humanbody showing the treatment of a tumor located in bladder with lightemitted by a fiber optic device, according to still another embodimentof the invention;

FIG. 5 is a diagrammatic illustration of the circulation system andbrain of the human head showing tumors located in various areas of thebrain and the treatment of one of the tumors with light emitted by afiber optic device, according to yet another embodiment of theinvention;

FIG. 6 is a diagrammatic illustration of the lungs and trachea of thehuman respiratory system showing a tumor located in one of the lungs andthe treatment of the tumor with light emitted by a fiber optic device,according to still another embodiment of the invention;

FIG. 7 is a diagrammatic illustration of the human circulatory system inthe upper portion of the body;

FIG. 8 illustrates a schematic diagram of a cancer treatment andevaluation system, according to an embodiment of the invention;

FIG. 9 illustrates a schematic diagram of a cancer treatment and imagingsystem, according to another embodiment of the invention;

FIG. 10 illustrates a schematic diagram of a cancer treatment system,according to yet another embodiment of the invention, wherein a thyroidtumor is being treated;

FIG. 11 illustrates a schematic diagram of a cancer treatment andimaging system, according to still another embodiment of the invention,wherein a thyroid tumor is being treated and imaged; and

FIG. 12 illustrates a schematic diagram of a cancer treatment andimaging system, according to yet another embodiment of the invention,wherein a thyroid tumor is being treated and imaged.

Various factors may lead one to suspect the presence of a smallcancerous or neoplastic tumor in a patient. Such factors include thepatient's genetic history, environmental conditions to which the patientis or has been exposed, the presence of biomarkers in the patient'sblood, or the presence of a lesion on a patient's skin or mucosalsurface. A small neoplasm of 1 to 2 millimeters (mm) in diameter,however, is often not recognized unless and until it produces someclinical symptom.

In a patient having a genetic mutation indicating a predisposition tocancer, prophylactic surgical intervention, such as a bilateralmastectomy performed in a patient having a genetic mutation indicating apredisposition to breast cancer, is seldom performed. Additionally, agenetic predisposition to one type of cancer may not lead to that typeof cancer, e.g. breast cancer, but it may lead to another unsuspectedtype of cancer, e.g. malignant melanoma. Even if the other type ofcancer is suspected, because of the finding of biomarkers in the blood,a small internal lesion may not be seen on radiography, or may not beaccessible by surgery, or the collateral complications may not beacceptable. It may not suffice to just know the biomarker for a tumor,because this information may not indicate whether the tumor is a primarysite or a metastatic site, the tissue of its origin, and/or itslocation. It is appreciated that some treatment techniques such assurgery or radiation may be useful, but only if the tumor is tissuespecific. Radiation and chemotherapy also have their own side effects,and may not destroy the tumor completely. Larger tumors present a muchcomplex problem, e.g., mutations in one area of the tumor are usuallydifferent from mutations in another area of the same tumor.

It is clearly preferable, then, to manage small early neoplasms thathave not progressed to a larger tumor to provide the patient an improvedclinical prognosis.

The invention includes a method of therapy for a non-surgicallyaccessible tumor by administering a combination of thermotherapy andimmunotherapy combined with gene delivery. In one or more embodiments,the gene delivery may use CRISPR-cas9 mediated Homology-IndependentTargeted Integration (HITI) or Homology Directed Repair (HDR). Thecombination therapy beneficially treats the tumor and prevents tumorrecurrence, either locally or at a different site, by boosting thepatient's immune response both at the time or original therapy and/orfor later therapy as a “booster” vaccine with or without viral-likeparticles (VLP) or adjuvants to the original therapy by administeringthem with antibody coated nanoparticles conjugated with checkpointinhibitors, such as PD-1, PD-L1, CTLA-4, Jagged 1 inhibitor 15D11, etc.and anti-inflammatory agents, such as Rock inhibitors, Fasudil, etc.,Wnt inhibitors, such as niclosamide etc. to enhance cellular immuneresponse of the patient while reducing the inflammatory response andpreventing an auto immune reaction or cytokine storm. In one embodiment,the vaccine with or without VLP, adjuvants, sodium bicarbonate to modifyacidic tumor cell environment is combined with checkpoint inhibitors,such as PD-1, PD-L1, CTLA-4, Jagged 1 inhibitor 15D11, etc., anti-VEGF,and anti-inflammatory agents, Rock inhibitors, such as Fasudil or Botox,or Wnt inhibitor, such as niclosamide, ivermectin and Selamectin, etc.,can be injected every six months or once a year, or is used in thetreatment of recurrent metastatic disease. In another embodiment theantibody coated nanoparticles or solid lipid nanoparticles are coatedwith thermosensitive polymers which are released at the temperature or41-42 C under thermotherapy and administered with checkpoint inhibitors,such as PD-1, PD-L1, CTLA-4, Jagged 1 inhibitor 15D11, etc. and Rockinhibitors, such as Fasudil or Botox, or Wnt inhibitor, such asniclosamide, ivermectin, or Selamectin and/or an antineoplasticmedication depending on the cancer, at lower dose than is normallyrecommended, but is made more effective by thermotherapy.

In one embodiment, N-myristoyltransferase (NMT) in cell cycle isinhibited which is involved in cell proliferation etc. NMT conjugateswith rare fatty acid and proteins. Lipid modification through inhibitionof N-myristoylation interferes with cell multiplication. Numerousantimalarial, antifungal, antiparasitic, antinematodes or antifilarial,antiviral, compounds can interfere with NMT function, but specificallythe compound IMP-1088 which inhibits capsid formation in viruses.However combining antibody coated nanoparticles with the compoundIMP-1088, NMT inhibitors, DDD85646 and DDD100870 and cHSC70 and/orenolase can be used alone or synergistically with Wnt inhibitors or Rockinhibitors, as anticancer therapeutic agent(s) conjugated with antibodycoated nanoparticles/medication injected intravenously orintra-arterially to the organ containing the tumor to inhibit the cancercell proliferation along with controlled thermotherapy.

In one embodiment the diagnosis and precision thermo-immune therapy isperformed locally or limited to an organ (e.g. head, lung, intestinaltract or extremities etc.) having radiographically or thermoacousticallyimaged a small tumor, anticancer therapeutic agent conjugated withantibody coated nanoparticles/medication is injected intravenously orintra-arterially to the organ containing the tumor, while sensitizingthe immune system to the tumor antigen to damage the original tumor andalso attack the potentially existing circulating tumor cells, tumorexosomes, or yet invisible metastatic lesion using thermotherapy locallycombined with antibody coated nanoparticles, checkpoint inhibitors, andan immune stimulator such as VLP, adjuvants, or toll like receptors, TNFalpha, etc. attached to antibody coated pluralities of nanoparticles orsolid lipid nanoparticles administered simultaneously to stimulatebody's immune response and eliminate the localized tumor and itscirculating cells, exosomes or metastatic lesions by innate immune cellsstimulation.

In one embodiment, combination antibody coated nanoparticles, such asnanoparticles or solid lipid or liposomes, dextrin, etc. conjugated withWnt inhibitors, such as Ivermectin and Selamectin and inhibitor ofN-myristoyltransferase, specifically IMP-1088, or NMT inhibitors,DDD85646 and DDD100870, checkpoint inhibitors and an immune stimulant(e.g., VLP), and sodium bicarbonate to modify acidic tumor cellenvironment, adjuvants, etc. administered intravenously orintra-arterially to work synergistically in preventing cancer cellproliferation of a small locally confined tumor along with controlledthermotherapy at the temperature of 40-43 degrees C. and treatsimultaneous localized immune therapy.

In one embodiment, early stage tumor therapy involves the anticancertherapeutic agents conjugated with antibody coatednanoparticles/medication injected intravenously or intra-arterially tothe organ containing the tumor while the thermotherapy performed with alaser fiber optic locally is brought to the tumor site through thefeeding artery, or a focused ultrasound is used in a thermal ornon-thermal mode from outside the body under temperature control usingan imaging system or thermoacoustic temperature imaging unit.

In one embodiment, a portable photoacoustic or thermoacoustic transducerprobe can be moved in any direction on the body, e.g., up and down, sideto side, etc., over the skin while emitting a laser light, microwave, orfocused ultrasound etc. and recording the sound waves from the antibodycoated nanoparticles producing a thermal expansion of nanoparticles.Using a processor in the photoacoustic or thermoacoustic unit, one usesthe photoacoustic or thermoacoustic response data to construct a two- orthree-dimensional image of a tumor for diagnosis of a lesion in thebody.

In one embodiment, the hand held photoacoustic/thermoacoustic probepermits scanning any bodily surface locally to diagnose presence of atumor, including but not limited to, the skin, breast, eye, centralnervous system (CNS), spinal cord, extremities, internal organs, lung,nose, chest, trachea, throat, abdomen, and urogenital organs afteradministration of the pluralities of antibody coated nanoparticles withcell penetrating peptides (CPP), activating CPP (ACPP), biotin, and/orstreptavidin or antibody coated nanoparticles conjugated with(alpha)-cyclodextrin, (beta)-cyclodextrin, (gamma)-cyclodextrin,hydroxypropyl-b-cyclodextrin (bHPCD) or solid lipid nanoparticlesinjected intravenously, topically or intra-arterially, and irradiatedwith a source of thermal energy such as laser, focused ultrasound or analternating magnetic field. The cyclodextrin nanoparticles may carrymedication, checkpoint inhibitors, VLP, adjuvants, and/or sodiumbicarbonate to modify the acidic tumor cell environment and act as slowrelease polymers at the tumor site, which can be released by thermalenergy.

In one embodiment, a laser fiber optic can be used for diagnosis andtherapy of the surface or internal lesions after administration of thepluralities of antibody-coated nanoparticles with cell penetratingpeptides (CPP), activating CPP (ACPP), biotin, streptavidin or antibodycoated nanoparticles conjugated with medication and(alpha)-cyclodextrin, (beta)-cyclodextrin, (gamma)-cyclodextrin,hydroxypropyl-b-cyclodextrin (bHPCD) or solid lipid nanoparticlesintravenously, topically for surface tumors, skin, mucosa, or throughthe accessible cavities such as the eyes, vitreous cavity, bladder,mouth, throat, esophagus, stomach, duodenum, rectum, colon, smallintestinal tract, and lung. In this embodiment, theantibody/cyclodextrin coated nanoparticles carry medication, such asRock inhibitors, Wnt inhibitors and/or Inhibitors ofN-myristoyltransferase, specifically IMP-1088, or NMT inhibitors,DDD85646 and DDD100870 and checkpoint inhibitors, or VLP, adjuvants,sodium bicarbonate to modify the acidic tumor cell environment andimmune stimulators and act as slow release polymers delivered via aflexible fiber optic and a tube is inserted through the natural orificesof the body (see FIGS. 1 and 4) or through a feeding artery or vein (seeFIG. 5) to bring the laser pulses close to the tissue containing atumor, and deliver the nanoparticles, but also use the laser energy toheat the accessible lesion or apply thermal energy externally usingmicrowave, focused ultrasound in thermal and non-thermal modes releasingthe medication(s) and stimulate the immune system to attack the tumorcells locally or wherever they are systemically by cellular immuneresponse. In one embodiment, the methods of delivery of antibody coatednanoparticles/medications are internal through the arteries or veins orthrough the body orifices as shown by the drawings (see FIGS. 1 and 4-7)or through an incision (e.g., made in the abdomen, etc. or subcutaneousor intramuscular, etc. injection in the desired location or tissue, orby topical application).

For example, as shown in FIG. 1, one laser fiber optic device 12 may beinserted through the mouth 16 of a person 10 so as to treat anesophageal tumor 28 by emitting light pulses generated a light source14. In addition, the laser fiber optic device 12 may be further insertedinto the esophagus 18 so as treat another tumor 30 located further downin the esophagus 18. In addition, as shown in FIG. 1, a skin tumor 32located near the right shoulder of the person 10 may be treated with alaser fiber optic device 34 having a light source 36. Next, turning tothe digestive system in FIG. 1, which includes the stomach 20, duodenum22, large intestine 24, and small intestine 26, further aspects of theillustrated embodiment will be described. In FIG. 1, it can be seen thata tumor 42 is disposed in the stomach 20, and another tumor 44 isdisposed in the large intestine 24. The tumor 44 in the large intestine24 is being treated with a laser fiber optic device 46 that has beeninserted into the large intestine 24 through the rectum. The laser fiberoptic device 46 has a light source 48. Also, as shown in FIG. 1, aphotoacoustic receiver 38 with cord 40 may be placed against the body ofthe person 10 in order to record a photoacoustic/thermoacoustic responseresulting from the thermal expansion of nanoparticles attached to thetumor (e.g., attached to tumor 42 in stomach 20) that are heated by thelaser light pulses from a fiber optic device. As described above, thenanoparticles may be delivered to the tumor site prior to the heatingthereof by a tube attached to the fiber optic device.

In one embodiment, the thermotherapy is done either internally orexternally using a laser applied through a fiber optic, etc. (see e.g.,FIGS. 1-6). In one embodiment, the antibody coatednanoparticles/medications are delivered intra-arterially orintravenously, but the thermotherapy is done externally using focusedultrasound, microwaves, radio frequency (RF), or using an alternatingmagnetic field when nanoparticles are magnetic or paramagnetic.

In some situations, as in the brain, the thermotherapy can be done at alow temperature (e.g., with focused ultrasound) that make the tumorvessels leakier prior to the injection on the antibody-coatednanoparticles. In another embodiment, the nanoparticles are injectedprior to the thermotherapy, etc.

In one embodiment, the source of energy is a focused ultrasound in anon-thermal or thermal mode applied from the outside the body while thepluralities of antibody-coated nanoparticles/medications arepiezoelectric, such as quartz, graphene, or perovskites, nanobubbles,perfluorocarbon liquid filled vesicles, etc., and administeredintra-arterially or intravenously, etc.

In one embodiment, as shown in the embodiment of FIG. 5, the delivery ofantibody coated nanoparticles/medication is done through an artery andthermotherapy is done with an internal laser using a fiber optic.

In one embodiment, one can deliver the antibody coatednanoparticles/medication internally and heat up the tumor preferablyexternally (e.g., by using a focused ultrasound). This is preferred forinternally located tumors, such as in the brain and internal organs thatcannot be reached by the laser or the size of the tumors are mostlylarger than 4 millimeters (mm) in diameter.

In another embodiment, any method or delivery of nanoparticles can beused depending on the location of the tumor and its thickness. Lasersare used mostly for small accessible lesions or superficial tumors, orlaser accessible tumors via a fiber optic or external laser for tumorshaving a thickness of about 1-4 mm. All other tumors can be preferablytreated by a source of energy that penetrates deep in the tissue (e.g.,focused ultrasound, microwaves, radio frequency (RF), or alternatingmagnetic field). In all cases, thermal delivery and temperature arecontrolled by either a photoacoustic imaging or thermoacoustic imagingunit to the desired level of temperature which is predetermined.

In one embodiment, a miniature capsule with an imaging camera, which isequipped with a laser system, is swallowed by the patient thatconstantly radiates a laser pulse, as it passes through the intestinaltract and transmits recorded images to a receiver outside the body.

In one embodiment, a photoacoustic sound wave is produced whenpluralities of antibody-coated nanoparticles are injected intravenouslywith cell penetrating peptides (CPP), activating CPP (ACPP), biotin,streptavidin, and/or the cyclodextrin antibody-coated nanoparticlescarry medication, inhibition of N-myristoyltransferase such as many antimalaria parasites medication, specifically IMP-1088, or NMT inhibitors,DDD85646 and DDD100870, checkpoint inhibitors, or VLP, adjuvants, sodiumbicarbonate to modify the acidic tumor cell environment and immunestimulators, such as TNF alpha, toxin, etc., and act as slow releasepolymeric drug release, where the nanoparticles accumulate at the siteof the tumor(s) in the intestinal tract and are stimulated with thelaser pulses which heat and create a photoacoustic sound that isrecorded by a receiver located outside the body and in contact with bodysurface, such as over the abdomen, etc., and is analyzed by softwarewhile an internal capsule with a video camera is traveling through theintestine and locates the presence of a tumor.

In one embodiment, as the capsule passes in front of a lesion in theintestinal tract, which has accumulated the pluralities of antibodycoated nanoparticles or solid lipid nanoparticles thereon, the laserpulse creates a photoacoustic sound that can be recorded by a receiverpositioned on the trunk of the patient and records the image of thelesion and the temperature at that site as the capsule travels throughthe intestine.

In one embodiment, the photoacoustic sound can be correlated with thevideo taken by the capsule and the location of the tumor is determinedeven if the tumor is too small to be recognized by CT scan orradiography or too small to make any visible or physical symptom. In oneembodiment, the capsule emits a significant amount of energy to increasethe temperature at the tumor site to release the medication, gene, fromthe antibody-coated nanoparticles with cell penetrating peptides (CPP),activating CPP (ACPP), biotin, streptavidin or the cyclodextrinantibody-coated nanoparticles which carry medication, such as Rockinhibitors, Wnt inhibitors or inhibitors of N-myristoyltransferase,IMP-1088, NMT inhibitors, DDD85646 and DDD100870, and checkpointinhibitors, and/or VLP, adjuvants, sodium bicarbonate to modify acidictumor cell environment and other immune stimulators, such as TNF alpha,IL 2 IL-6, toll like receptor 7/8 that stimulate innate immune cellresponse, etc. and are released from the slow release polymers, anddamage and kill the tumor cells while releasing their cellular antigensin the circulation to attract cellular immune response and kill theremaining tumor cells that are present locally or might exist in thebody.

In one embodiment, a laser fiber optic with or without the camera, whilepulsing laser energy, is passed through the mouth to the stomach orthrough the rectum into the colon or through the ureter inside thebladder, through the mouth, throat, trachea, and bronchi, etc. orthrough the vagina inside the uterus or further through one of thefallopian tubes toward the ovaries.

In one embodiment, the laser pulse produces a photoacoustic orthermoacoustic response from the pluralities of antibody-coatednanoparticles with cell penetrating peptides (CPP), activating CPP(ACPP), biotin, streptavidin or the cyclodextrin antibody coatednanoparticles carrying medication, such as Rock inhibitors, Wntinhibitors or inhibitors of N-myristoyltransferase, such as manyanti-malaria parasites medication, specifically IMP-1088 compound or NMTinhibitors, DDD85646 and DDD100870, and checkpoint inhibitors, or VLP,adjuvants, sodium bicarbonate to modify the acidic tumor cellenvironment and immune stimulators, such as TNF alpha, IL6, IL 17, tolllike receptors, etc. and act as slow release polymeric drug releasecarrying medication to be attached to the tumor cells injectedintravenously or intra-arterially 1-2 or more minutes ahead, permittingthe nanoparticles to travel in the body and attach to the tumor cellsthat can be exposed to laser radiation, focused ultrasound, microwave oran alternating magnetic field to heat the nanoparticles and to produce aphotoacoustic or thermoacoustic sound to be recorded by a photoacousticor thermoacoustic transducer (an ultrasound receiver) located on thesurface of the body to image the tumor while measuring the temperaturegenerated at the tumor site by the laser or other energy source, such asfocused ultrasound, microwave, or alternating magnetic field to imagethe temperature and the tumor in a 2-D and 3-D format, increasing thethermal radiation by a processor connected to the photoacoustic unit andthermal delivery unit to increase the tumor temperature and damage orkill the tumor cells at temperatures of 43 to 45-47 degrees C. andrelease cytoplasmic tumor antigens to attract dendritic cells, T-cells,and/or other killer cells to remove the tumor as they circulate in thebody destroying the circulating or sessile tumor cells elsewhere in thebody.

In one embodiment, where a tumor is inaccessible through the naturalorifices, a fiber optic endoscope 94 is inserted through a smallincision in the abdomen in the peritoneal cavity toward liver, spleen,pancreas, or kidney (see e.g., FIG. 3, which depicts a tumor 96 locatedin the pancreas 90) for diagnostic or therapeutic purposes using thelaser thermal energy, to recognize the location of the tumor afterinjecting intravenously the antibody-coated pluralities of nanoparticleswith cell penetrating peptides (CPP), activating CPP (ACPP), biotin,and/or streptavidin, or antibody-coated nanoparticles conjugated with(alpha)-cyclodextrin, (beta)-cyclodextrin, (gamma)-cyclodextrin,hydroxypropyl-b-cyclodextrin (bHPCD) conjugated with a thermosensitivepolymer carrying medication and/or a gene, medication or immunestimulators. In this embodiment, shining the laser light over thesuspected tumor/nanoparticle complex area creates a photoacoustic sound,which is imaged by a receiver or receivers located on the skin of theabdomen, then heating preferentially the antibody-coated pluralities ofnanoparticles attached to the tumor cells by a laser, microwaves, orfocused ultrasound, etc. damaging the tumor cells at a temperature of37-43 degrees C., thereby releasing the conjugated medication, gene,toxins, Wnt inhibitors, or Rock inhibitors along with immune stimulatorsto kill and eliminate the cancer cells. In FIG. 3, several digestiveorgans of the human body are illustrated around the area of the pancreas90 being treated by the fiber optic device 94, namely the gallbladder82, liver 84, and duodenum 85. Also, illustrated in FIG. 3 are the bileduct 86, the pancreatic duct 88, and the opening 92 of the bile andpancreatic ducts 86, 88 into the duodenum 85.

In one embodiment, other tumors inside the body can be accessed throughinsertion of a fiber optic through the blood vessels, arteries, or veinsof an organ (see e.g., FIG. 5) to induce a more organ specificdiagnosis, and thermo-immune therapy without affecting the normal cells(e.g., in the brain, eye, or extremities) for tumors localized in thehead and neck or urogenital organs, lung, etc.

In one embodiment, the thermotherapy is performed by injectingintravenously or intra-arterially, antibody or monoclonalantibody-coated pluralities of nanoparticles or solid lipidnanoparticles conjugated with thermosensitive polymers such aschitosan/medication or antibody coated nanoprticles conjugated with(alpha)-cyclodextrin, (beta)-cyclodextrin, (gamma)-cyclodextrin,hydroxypropyl-b-cyclodextrin (bHPCD, liposomes, solid lipidnanoparticles with nanowires, nanotubes, nanoshells, nanocages, periodicmesoporous organosilica nanoparticles conjugated with immunestimulator(s), such as interferon alpha, toll like receptors, IL 2, IL6,IL17, or VLP, adjuvants, sodium bicarbonate to modify the acidic tumorcell environment and CPP, with medication, inhibitors ofN-myristoyltransferase, such as many anti-malaria parasites medication,specifically IMP-1088, or NMT inhibitors, DDD85646 and DDD100870, andRock inhibitors, Wnt inhibitors which interferes with cellularproliferation or is injected through a major artery after insertion of aflexible laser fiber optic with a tube or a needle in a few steps oftherapy: (a) administering the pluralities of antibody coatednanoparticles or liposomes, solid lipid nanoparticles, etc.; (b)applying thermal energy to the lesion and imaging it by a thermoacousticunit to control the temperature to 40-43 degrees C., to release themedication from the nanoparticles or liposomes or solid lipidnanoparticles at the tumor site and irradiate the tumor cells withthermal energy; (c) continue administering antibody coatednanogel/nanoparticles, such as quantum dots or other nanoparticles suchas gold or silica, magnetic, non-magnetic or hydrogels or liposomes orsolid lipid nanoparticles (e.g., made of desired molar concentration andthe ANG nanogel content) with or without various concentrations of acrosslinker, fibrinogen, adenosine diphosphate (ADP) to convertthrombinogen to thrombin and the fibrinogen to fibrin locally at thesite of the tumor vasculature creating a precise localized vascularocclusion at the tumor site only while sparing the normal surroundingtissue.

In one embodiment, in contrast to the standard technique of embolizationof a large vessel supplying a tumor which leads to indiscriminateclosure of supply of a large area of an organ and damaging normal cells,the precise thermotherapy damages the endothelial cells and the tumorcells, releasing the medication from the antibody coated nanoparticles,namely releasing prothrombotic medication, checkpoint inhibitors, immunestimulators, anti-VEGF enhancing platelets binding and aggregation, andclotting; thus creating a discrete blood clot or thrombus, whichobstructs the blood supply to the tumor, and starves the tumor from itsblood supply while releasing the immune stimulating agents andmedications slowly from the polymeric nanoparticle compounds and providea long term local or systemic immunotherapy to the patient, andwithdrawing blood after therapy one obtains increased tumor biomarkersproving the presence of a tumor even it was not initially not visibleradiologically and administering antibody-coated pluralities ofnanoparticles coated with dimethylacetyl-beta-cyclodextrin to inhibitexcessive innate immune response locally and prevent excessive edema atthe tumors surrounding site.

In one embodiment, one creates a precise local temperature increase andvascular occlusion at the site of the tumor and tumor vessels regardlessof its location in the body and the size of the lesion, the injectedantibody or monoclonal antibody-coated pluralities of nanoparticlesconjugated with thermosensitive polymers, such as chitosan, and/ormedication or antibody-coated nanoparticles conjugated with(alpha)-cyclodextrin, (beta)-cyclodextrin, (gamma)-cyclodextrin,hydroxypropyl-b-cyclodextrin (bHPCD, liposomes or solid lipidnanoparticles containing nanowires, nanotubes, nanoshells, nanocages,periodic mesoporous organosilica nanoparticles with immunestimulator(s), such as interferon alpha, toll like receptors, IL 2, IL6,IL17 or VLP, adjuvants, sodium bicarbonate to modify the acidic tumorcell environment and CPP, medication such as inhibitors ofN-myristoyltransferase, such as many anti-malaria parasites medication,specifically IMP-1088 or NMT inhibitors, DDD85646 and DDD100870, Rockinhibitors, Wnt inhibitors are given intravenously but preferablythrough a feeding artery after insertion of a flexible laser fiber opticwith a tube or just a small gauge needle in the vessel in differentsteps of therapy: (a) administering the pluralities of nanoparticles orcombined with liposomes or solid lipid nanoparticles, etc.; (b) applyingthermal energy to the lesion either with laser or focused ultrasound oran alternating magnetic field, microwave, etc. and using thermoacousticimaging to control the energy delivery system to a temperature of 40-43degrees C., to release the medication and damage the tumor cellsmembrane and the their endothelial cells membrane of their feedingvessels and capillaries supplying the tumor cells; (c) releasing fromthe antibody-coated nanogel/nanoparticles, such as quantum dots,liposomes, solid lipid nanoparticles, or other nanoparticles, such asgold or silica, magnetic, non-magnetic or hydrogels or liposomes orsolid lipid nanoparticles or cyclodextrin, e.g., coated with a desiredmolar concentration, fibrinogen, adenosine diphosphate (ADP) to releasethem and to convert locally thrombinogen to thrombin and the fibrinogento fibrin, producing a local thrombus locally at the site of the tumorvasculature, thus creating a precise vascular occlusion at the area ofthe tumor under observation. This is in contrast to the presently doneembolization of a large vessel that not only supplies a tumor but largeareas of an organ indiscriminatingly, such as in liver, brain, spleen,pancreas, lung, kidney, genitourinary system, or any other part of thebody.

In one embodiment, after intravenous injection of antibody-coatednanoparticles, a localized thermotherapy is performed that damages thetumor cells and the endothelial cells membrane enhancing plateletsbinding and creating a discrete blood clot or thrombus, which obstructsthe blood supply to the tumor precisely, and starves the tumor fromoxygen and nutrition, while releasing from the nanoparticles, the innatebody's immune stimulating compound agents, such as antibody coatednanoparticles conjugated with interferon alpha, toll like receptors, IL2, IL6, IL17 and adjuvants and checkpoint inhibitors with CPP, anti-VEGFand medications, such as Rock inhibitors, Wnt inhibitors or inhibitionof N-myristoyltransferase which interferes with cellular proliferation,to be released slowly for a long time from the polymeric compounds ofthe nanoparticles and provide a long term local or systemicimmunotherapy to the patient followed with withdrawing blood aftertherapy to obtain tumor biomarkers for the future vaccine production.

In one embodiment, the injection of antibody or monoclonal antibodycoated pluralities of nanoparticles conjugated with thermosensitivepolymers, such as chitosan, and/or medications and antibody-coatednanoparticles conjugated with (alpha)-cyclodextrin, (beta)-cyclodextrin,(gamma)-cyclodextrin, hydroxypropyl-b-cyclodextrin (bHPCD) withanti-VEGF, immune stimulator(s), such as interferon alpha, toll likereceptors, IL 2, IL6, IL17, or VLP, adjuvants, sodium bicarbonate tomodify the acidic tumor cell environment and CPP is done through anartery after insertion of a flexible laser fiber optic with a tube inthese different steps of therapy: (a) administering the pluralities ofantibody-coated nanoparticles carrying checkpoint inhibitors, along withimmune stimulators; (b) applying thermal energy to the lesion andsimultaneous thermal imaging to control the temperature to 40-43 degreesC., to release the medication from the coating of the nanoparticles anddamage and/or irritate the tumor cells membrane; (c) administeringantibody-coated platelets from the patient's blood, coating theplatelets with antibodies, or monoclonal antibodies and reintroducingthe coated platelets into the patient to attach to the tumor and itsassociated vascular supply, creating a blood clot or thrombus, whichobstructs the blood supply to the tumor, and nutritionally starves thetumor while the immune stimulating agents and medications continue to bereleased slowly for a long time from the polymeric compounds and providea long term local or systemic immunotherapy to the patient, which can bealso injected later in another body location to act as a vaccine.

In one embodiment, the thermotherapy of the injected antibody ormonoclonal antibody-coated pluralities of nanoparticles conjugated withthermosensitive polymers, such as chitosan, and/or medication withimmune stimulator(s) interferon alpha, toll like receptors, IL 2, IL 6,IL17, or VLP and CPP is done through an artery after insertion of alaser fiber optic with a tube in at least four different steps oftherapy: (a) administering the pluralities of nanoparticles, (b)applying thermal energy to the lesion and imaging to control thetemperature to 40-43 degrees C., to release the medication and irritatethe tumor cells, (c) administering antibody coatednanogel/nanoparticles, such as quantum dots, or other nanoparticles suchas gold or silica, magnetic, non-magnetic or hydrogel (e.g., made ofdesired molar concentration and the ANG nanogel content with or withoutvarious concentrations of a cross-linker) to attach to the tumor and itsassociated vascular supply, enhancing platelets binding and clotting,creating a blood clot or thrombus, which obstructs the blood supply tothe tumor, and nutritionally starves the tumor while the immunestimulating agents and medications continue to be released slowly for along time from the polymeric compounds and provide a long term local orsystemic immunotherapy to the patient, and (d) withdrawing blood aftertherapy for increased tumor biomarker support for the existence of atumor even it was not initially visible radiologically for creating avaccine that can be injected at a later time subcutaneously.

In one embodiment, injection of antibody or monoclonal antibody-coatedpluralities of nanoparticles conjugated with thermosensitive polymers,such as chitosan, and/or medication or antibody-coated nanoparticlesconjugated with (alpha)-cyclodextrin, (beta)-cyclodextrin,(gamma)-cyclodextrin, hydroxypropyl-b-cyclodextrin (bHPCD) with immunestimulator(s), such as interferon alpha, toll like receptors, IL 2, IL6,IL17, or VLP, adjuvants, sodium bicarbonate to modify the acidic tumorcell environment and CPP, Wnt inhibitors or Rock inhibitors, anti-VEGFis done through an artery after insertion of a flexible laser fiberinside a tube imaged during its insertion in an artery for immunethermotherapy by: (a) administering the pluralities of antibody coatednanoparticles; (b) applying external or internal laser thermal energy,focused ultrasound, microwave or an alternating magnetic field to thelesion and thermoacoustic imaging to control the temperature to 40-43degrees C., to release the medication and irritate the tumor cells andendothelial cells of the tumor; (c) administering antibody-coatednanogel/nanoparticles, such as quantum dots or other nanoparticles, suchas gold or silica, magnetic non-magnetic or hydrogel (e.g., made ofdesired molar concentration and the ANG nanogel content with or withoutvarious concentrations of a photosensitizer) to attach to the tumor andits associated vascular supply, enhancing platelets binding andclotting, creating a blood clot or thrombus, which obstructs the bloodsupply to the tumor, and starves the tumor from its nutrition and oxygenwhile releasing immune stimulating agents and medications slowly in acontinuous manner from the polymeric coated nanoparticles and providinga long term local or systemic immunotherapy to the patient; (d)withdrawing blood after therapy to obtain increased tumor biomarkersthat support the presence of a tumor even it was not initially visibleradiologically; and (e) administering antibody coated pluralities ofnanoparticles coated with dimethylacetyl-beta-cyclodextrin to inhibitexcessive innate immune response locally and prevent excessive edema atthe tumors surrounding site.

In one embodiment, the laser fiber optic is inside a flexible tubethrough which one injects pluralities of antibody-coated nanoparticleswith cell penetrating peptides (CPP), activating CPP (ACPP), biotin,and/or streptavidin or antibody-coated nanoparticles conjugated with(alpha)-cyclodextrin, (beta)-cyclodextrin, (gamma)-cyclodextrin,hydroxypropyl-b-cyclodextrin (bHPCD) conjugated with thermosensitivepolymers, such as PLA, PGA, chitosan, polyanhydride, polyanhydride,porous silicon carrying medication, siRNS, DNA, RNAi, CRISPR-cas9,inhibitors of N-myristoyltransferase, such as many anti-malariaparasites medication, specifically IMP-1088, NMT inhibitors, DDD85646and DDD100870, Wnt inhibitors, or Rock inhibitors or CAR-t cells grownin cell culture and sensitized to the tumor antigen along with immunestimulation with VLP, toll-like receptors, adjuvants, sodium bicarbonateto modify the acidic tumor cell environment, toll like receptor 7/8 orinterferons antibody-coated nanoparticles dendrimers, etc. can beinjected in the circulation locally, intra-arterially to attach to thetumor cells and damage their cell membranes with thermal energy, such aslaser, focused ultrasound, alternating magnetic field, or microwaves,etc. increasing the nanoparticles' and/or tumor cell's temperature to40-43 degrees C. or more, and releasing the medication, immunestimulator, locally at the tumor site and enhance local immune therapyfor a long duration and innate immune stimulation for a long period oftime due to the release of the immune stimulators from the nanoparticlesor porous silicone nano or microparticles, and withdrawing blood aftertherapy to measure increased tumor biomarkers and use them for vaccinetherapy.

In one embodiment, E-selectin coated pluralities of antibody-coatednanoparticles are injected intravenously with cell penetrating peptides(CPP), activating CPP (ACPP), or antibody-coated nanoparticlesconjugated with (alpha)-cyclodextrin, (beta)-cyclodextrin,(gamma)-cyclodextrin, hydroxypropyl-b-cyclodextrin (bHPCD), biotin,and/or streptavidin conjugated with medication and an Wnt inhibitorbinds to sialylated carbohydrates on the surface proteins of certainleukocytes, increasing the nanoparticle temperature using a source ofthermal energy to 40-43 degrees C. or more, and releasing the medicationlocally to the tumor cells and attracting neutrophils, monocytes,eosinophils, memory-effector T-like lymphocytes, and natural killercells to further damage and remove the tumor cells.

In one embodiment, the CAR-T cells or killer cells are grown in a tissueculture with antibody-coated nanoparticles with cell penetratingpeptides (CPP), activating CPP (ACPP), biotin, and/or streptavidin, orantibody-coated nanoparticles conjugated with (alpha)-cyclodextrin,(beta)-cyclodextrin, (gamma)-cyclodextrin, hydroxypropyl-b-cyclodextrin(bHPCD) which are conjugated with E-selectin to attach to the surface ofthe CAR-T cells and conjugated with VLP, adjuvants, sodium bicarbonateto modify the acidic tumor cell environment or other immune stimulatingagents conjugated with polymeric nanoparticles or porous siliconnanoparticles coated with slow release polymers of PAL, GA, PLGA, orchitosan administered before and after thermotherapy to attach to thetumor cells and enhance cellular immune response after thermotherapy for2 to 3 months or more as long as the slow release polymers last in thebody.

In one embodiment, the CAR-T cells or killer cells are in grown tissueculture with antibody-coated nanoparticles with cell penetratingpeptides (CPP), activating CPP (ACPP), biotin, and/or streptavidin, or(alpha)-cyclodextrin, (beta)-cyclodextrin, (gamma)-cyclodextrin,hydroxypropyl-b-cyclodextrin (bHPCD) which are conjugated withE-selectin to attach to the surface of the CAR-T cells and amedications, toxins, enzymes, TNF, TRAIL, VLP, adjuvants, sodiumbicarbonate to modify the acidic tumor cell environment, toxins,propranolol, a beta blocker, or an anti-VEGF to inhibit the tumor'svascular growth and can be injected intra-arterially at less than 1/10-1/50th of the quantities used systemically, through laser fiber optictube slowly before and after thermotherapy of a localized tumor, to bereleased slowly to attack repeatedly, the tumor cells in the specificorgan, and induce a localized lasting immune response or induce animmune response in the body to eliminate potential existing tumor cellsor invisible metastasis and autoimmune response.

In one embodiment, the release of antibody-coated pluralities ofnanoparticles with cell penetrating peptides (CPP), activating CPP(ACPP), biotin, and/or streptavidin or antibody-coated(alpha)-cyclodextrin, (beta)-cyclodextrin, (gamma)-cyclodextrin,hydroxypropyl-b-cyclodextrin (bHPCD) are observed after intra-arterialinjection with an imaging system such as MRI, or ultrasound to verifythe position of the tumor that is being treated with controlledthermotherapy using electromagnetic radiation, microwave, radiofrequency(RF), or focused ultrasound or alternating magnetic field or an electricfield, and the lesion is imaged by a photoacoustic or thermoacousticimaging system, the temperature is controlled by the thermoacoustic unitconnected via a processor to the thermal delivery system to controlthermal energy delivery, and prevent over cooking of the tissue via thethermoacoustic imaging system that measures the temperature rise 100times per second and is in communication with the thermal energydelivery device so that the desired temperature of, for example, 41-43degrees C. is achieved or maintained for a preferred time.

In one embodiment, the laser fiber optic with the tube is insertedthrough the carotid artery to reach either sides of the CNS harboring atumor, such as glioblastoma, etc. to release pluralities ofantibody-coated nanoparticles with cell penetrating peptides (CPP),activating CPP (ACPP), biotin, and/or streptavidin, Wnt or Rockinhibitors or antibody coated (alpha)-cyclodextrin, (beta)-cyclodextrin,(gamma)-cyclodextrin, hydroxypropyl-b-cyclodextrin (bHPCD) conjugatedwith polymers, such as PLA or PGA, nanoparticles of porous silicon,chitosan attached with medication or immune stimulators in thecirculation so that the majorities of nanoparticles reach at a highconcentration at the tumor site and are released as needed byapplication of internal or external thermal energy such as laser,focused ultrasound, alternating magnetic field, etc. under observationof the tumor's temperature kept at 41-43 degrees C. for a desired timewhile the thermal energy delivery system is controlled via a processorthat connects the thermoacoustic imaging unit that measures and recordsto the thermal energy delivery unit so that the predeterminedtemperature is achieved inside the tumor and maintained for a desiredtime to prevent over-cooking of the normal tissue.

In one embodiment, the laser fiber optic with the tube is insertedthrough the femoral artery through the abdomen and moved toward anyorgan, such as the kidney, intestine, spleen, liver, heart, lung orreaches the carotid artery or any other part of the brain, to releasepluralities of antibody-coated nanoparticles in the circulation so thatthe nanoparticles/medications reach a higher concentration at the tumorsite than would be reached when injected intravenously where most of thenanoparticles/medications are taken up by the liver and spleen'sreticuloendothelial cells before reaching the tumor site.

In one embodiment, the laser fiber optic with the tube is insertedthrough the femoral or radial/femoral artery (see FIG. 7) to reach thetumor in the bone or extremities to release the pluralities ofantibody-coated nanoparticles/medication/propranolol with cellpenetrating peptides (CPP), activating CPP (ACPP), biotin, and/orstreptavidin or antibody coated nanoparticles conjugated with(alpha)-cyclodextrin, (beta)-cyclodextrin, (gamma)-cyclodextrin,hydroxypropyl-b-cyclodextrin (bHPCD) conjugated with thermosensitivepolymers such as PLA, PGA, PLGA, chitosan, polyanhydride orantibody-coated nanoparticles/microparticles of porous silicon carryingmedication, siRNS, DNA, RNAi, CRISPR-Cas9, Wnt inhibitors, or Rockinhibitors or CAR-t cells grown in cell culture and sensitized to thetumor antigen along with immune stimulation with VLP, adjuvants, sodiumbicarbonate to modify the acidic tumor cell environment, toll likereceptors 7/8 or interferons, TNF alpha, IL2 can be injected in thecirculation locally to attach to the tumor cells so as to then damagethem with thermal energy such as laser, focused ultrasound, alternatingmagnetic field, or microwaves by increasing the nanoparticlestemperature to 40-43 degrees C. or more and releasing the medication,and propranolol locally to the tumor cells.

The human circulatory system 144 is represented in diagrammatic form inFIG. 7. In FIG. 7, it can be seen that the circulatory system 144includes left and right common carotid arteries 146, 150, left and rightinternal jugular veins 148, 152, a right brachial artery 154, a rightvein 156, an abdominal aorta artery 158, an inferior vena cava 160, aleft femoral artery 162, and a left common iliac vein 164. As describedabove, in one or more embodiments, the laser fiber optic with the tubecarrying the antibody-coated nanoparticles may be inserted through oneof the arteries or veins 146-164 depicted in FIG. 7.

In one embodiment, the flexible laser fiber optic with the tube isinserted through the radial arteries, to reach the lung or the heart todeliver pluralities of antibody-coated nanoparticles with cellpenetrating peptides (CPP), activating CPP (ACPP), biotin, and/orstreptavidin or antibody coated nanoparticles conjugated with(alpha)-cyclodextrin, (beta)-cyclodextrin, (gamma)-cyclodextrin,hydroxypropyl-b-cyclodextrin (bHPCD) conjugated with thermosensitivepolymers, such as PLA PGA, chitosan, polyanhydride carrying medication,siRNS, DNA, RNAi, CRISPR-Cas9, Wnt inhibitors, or Rock inhibitors orCAR-t cells grown in cell culture and sensitized to the tumor antigenalong with immune stimulation with VLP, adjuvants, sodium bicarbonate tomodify the acidic tumor cell environment, toll-like receptors 7/8 orinterferons can be injected in the circulation locally to attach to thetumor cells so as to then damage them with thermal energy such as laser,focused ultrasound, or alternating magnetic field by increasing thenanoparticles temperature to 40-43 degrees C. or more and releasing themedication locally to the tumor cells.

In one embodiment, for example, a brain tumor is located in the right orleft temporal lobe of the brain, and the laser fiber/tube is insertedthrough the carotid artery (see FIG. 5) for delivery of pluralities ofantibody-coated nanoparticles/medication with cell penetrating peptides(CPP), activating CPP (ACPP), biotin, and/or streptavidin or antibodycoated nanoparticles conjugated with (alpha)-cyclodextrin,(beta)-cyclodextrin, (gamma)-cyclodextrin, hydroxypropyl-b-cyclodextrin(bHPCD) conjugated with thermosensitive polymers such as PLA, PGA,chitosan, and polyanhydride or porous silicon nanoparticles and/ormicroparticles carrying medication, siRNS, DNA, RNAi, CRISPR-Cas9, Wntinhibitors, checkpoint inhibitors, or Rock inhibitors or CAR-t cellsgrown in cell culture and sensitized to the tumor antigen along withimmune stimulation with VLP, adjuvants, sodium bicarbonate to modify theacidic tumor cell environment, toll-like receptors 7/8 or interferons,the antibody-coated nanoparticles can be injected in the circulationlocally to attach to the tumor cells so as to then damage them withthermal energy, such as laser, focused ultrasound, or alternatingmagnetic field, thereby increasing the nanoparticles temperature to40-43 degrees C. or more and releasing the medication locally to thetumor cells and damage the tumor cells and combining it with humoral andcellular immune therapy, while a drainage tube is placed in the jugularvein of the right or left side to collect cellular debris and toxins andpass it through a dialysis system and return the cleansed blood back tothe patient eliminating the adverse effects of chemotherapy and immunetherapy to the patient, thus creating a concept of lasting therapeuticintervention with potential of ease of re-injection as vaccine andre-stimulation of the immune system.

For example, as shown in FIG. 5, a laser fiber optic device 124 may beinserted through the internal carotid artery 120 in the head of a personso as to treat a tumor 118 in the brain 112 of the person by emittinglight pulses generated a light source 126. Also, as shown in FIG. 5,additional tumors 114, 116 are located in the brain 112 of the person.The additional tumors 114, 116 may also be treated using a laser fiberoptic device inserted through a nearby artery in the head of the person.As one example, a fiber optic device may be inserted through theexternal carotid artery 122 in the head of the person so as to treat thetumor 116. Also, as shown in FIG. 5, a photoacoustic receiver 128 withcord 130 may be placed against the head of the person in order to recorda photoacoustic/thermoacoustic response resulting from the thermalexpansion of nanoparticles attached to the tumor (e.g., attached totumor 118 in the brain 112) that are heated by the laser light pulsesfrom the fiber optic device 124. The nanoparticles may be delivered tothe tumor site prior to the heating thereof by a tube attached to thefiber optic device 124.

In one embodiment, to prevent a severe autoimmune response after tumorimmunotherapy, one uses the return blood, for example, from the jugularvein for extracorporeal plasmapheresis, the nanoparticle assistedthermotherapy and imaging system to apply heavy thermal energy to a tubecontaining blood cells and to achieve a temperature as high as 60degrees C. to kill the sensitized immune cells containing nanoparticles.Blood is then passed through a dielectrophoresis system to characterizeand remove dead or live T-cells, sensitized killer cells, and tumorcells prior to re-infusing blood in the patient while simultaneouslyadministering antibody-coated nanoparticles conjugated withanti-inflammatory agents, including biologics and mycophenolic acid toreduce the severe autoimmune response often seen after tumorimmunotherapy.

In one embodiment, the pluralities of antibody-coated nanoparticles ordendrimers with cell penetrating peptides (CPP), activating CPP (ACPP),biotin, and/or streptavidin or antibody-coated nanoparticles conjugatedwith (alpha)-cyclodextrin, (beta)-cyclodextrin, (gamma)-cyclodextrin,hydroxypropyl-b-cyclodextrin (bHPCD) conjugated with thermosensitivepolymers such as PLA, PGA, PLGA, chitosan, polyanhydride, anhydride orporous silicon nanoparticles and/or microparticles carrying medicationand immune stimulators, siRNS, DNA, RNAi, to modify the genetic mutationof the tumor cells using CRISPR-Cas9 to reduce the expression ofcheckpoint proteins without the use of a viral vector, and can beinjected intravenously or preferably intra-arterially in the circulationat a dose far below the systemically used non-toxic dose so that thenanoparticles travel and attach to the tumor cells directly in an organand eliminate the tumor cells or modify their genetic component withoutusing a vector using controlled thermotherapy and using CRISPR-Cas9mediated Homology-Independent Targeted Integration (HITI) or HomologyDirected Repair (HDR).

In one embodiment, the pluralities of antibody-coated nanoparticles withcell penetrating peptides (CPP), activating CPP (ACPP), biotin, and/orstreptavidin or antibody-coated nanoparticles/medication conjugated with(alpha)-cyclodextrin, (beta)-cyclodextrin, (gamma)-cyclodextrin,hydroxypropyl-b-cyclodextrin (bHPCD) conjugated with thermosensitivepolymers, such as PLA, PGA, PLGA, chitosan, polyanhydride, anhydride orporous silicon nanoparticles and/or microparticles carrying medication,siRNS, DNA, RNAi to modify the genetic mutation of the tumor cells usingCRISPR-Cas9 to reduce the expression of checkpoint proteins without theuse of a viral vector but combined with Wnt inhibitors, or Rockinhibitors/antibody-coated nanoparticles can be injected intravenouslyor preferably intra-arterially in the circulation at a dose far belowthe systemically non-toxic dose or less than 1/100 the non-toxic doseused systemically so that the nanoparticles travel directly to the tumorcells locally, attach to them in an organ and eliminate the tumor cellsor modify their genetic component by using CRISPR-Cas9 mediatedHomology-Independent Targeted Integration (HITI) or Homology DirectedRepair (HDR) component without using a viral vector.

In one embodiment, the pluralities of magnetic or paramagnetic coatednanoparticles, or magnetic luminescent porous SI nanoparticles and/ormicroparticles with cell penetrating peptides (CPP), activating CPP(ACPP), biotin, and/or streptavidin or antibody-coated nanoparticlesconjugated with (alpha)-cyclodextrin, (beta)-cyclodextrin,(gamma)-cyclodextrin, hydroxypropyl-b-cyclodextrin (bHPCD) areadministered intra-arterially and heated at the tumor site either withthe laser light internally with the use of a fiber optic or from outsidewith a focused ultrasound in a compressive non-thermal focused mode tostrip from the nanoparticles, the coating conjugated with a gene,medication or Wnt or Rock inhibitors, and then heated to 40-43 degreesC. with the thermal mode of a focused ultrasound, microwaves, or lasercreating a thermal effect on the tumor while the degree of thetemperature is imaged using a photoacoustic or thermoacoustic thermalimaging system where the receiver is attached to the surface of theskull, neck, or body elsewhere or the use of an MRI to locate the tumorand measure the tumor/nanoparticle temperature. The control of thetemperature is achieved with an operative connection between thethermoacoustic unit and the thermal delivery unit (e.g., a hardware andsoftware-based connection), which controls the thermal energy dependingon what temperature is needed and dialed on the system.

In one embodiment, the thermal energy is provided with either analternating magnetic field or a microwave unit of an RF unit or focusedultrasound while the thermoacoustic system controls the desired energylevel and duration with a hardware/software-based connection to thermaldelivery system.

In one embodiment the tumor/nanoparticles are heated to the temperatureof 37-40 degrees C. or 40-43 degrees C. and maintained for 1 second to10 minutes as needed to damage the tumor cells and release themedication.

In one embodiment, one uses the laser fiber optic/tube to induce alocalized immunotherapy by administering pluralities of antibody-coatednanoparticles with cell penetrating peptides (CPP), activating CPP(ACPP), biotin, and/or streptavidin or antibody coated nanoparticlesconjugated with (alpha)-cyclodextrin, (beta)-cyclodextrin,(gamma)-cyclodextrin, hydroxypropyl-b-cyclodextrin (bHPCD) conjugatedwith checkpoint inhibitors, and or monoclonal antibodies or aptamers, orimmune stimulators with or without injection of a limited number ofCAR-T cells to phagocytize the damaged tumor cells.

In one embodiment, the CAR-T cells are modified with RNAi or sRNA, etc.in vitro to silence the immune checkpoints in them so that after theiradministration they do not respond to the tumor cell production ofcheckpoint inhibitors and attack the tumor cells where they find them.

In one embodiment the pluralities of antibody coated nanoparticles withcell penetrating peptides (CPP), activating CPP (ACPP), biotin, and/orstreptavidin or antibody coated nanoparticles conjugated with(alpha)-cyclodextrin, (beta)-cyclodextrin, (gamma)-cyclodextrin,hydroxypropyl-b-cyclodextrin (bHPCD) are conjugated with viral-likeparticles (VLP), adjuvants or toxin, viruses not only to damage thetumor cells but induce localized immune response, inflammation toattract the patient's lymphocytes, and killer cells to remove the deadtumor cells and provide systemic immunity to circulating tumor cells.

In one embodiment, the blood returning from the brain, etc. where thetumor is located is withdrawn through the jugular vein, passed through adialysis or dielectrophoresis system to clean the blood from the deadcells and remove checkpoint inhibitors, the VLP, adjuvants, or othertoxins produced by the dead tumor cells to prevent a cytokine storm.

In one embodiment, after the thermo-immune therapy, Wnt inhibitors, orRock inhibitors are administered to the tumor by conjugating them withpluralities of antibody-coated nanoparticles/medication, with cellpenetrating peptides (CPP), activating CPP (ACPP), biotin, and/orstreptavidin or antibody coated nanoparticles conjugated withnanoparticles coated with (alpha)-cyclodextrin, (beta)-cyclodextrin,(gamma)-cyclodextrin, hydroxypropyl-b-cyclodextrin (bHPCD).Administering antibody coated dimethylacetyl-beta-cyclodextrin aftertherapy inhibits excessive innate immune response and prevent excessiveedema at the tumors surrounding site, to reach the tumor area andprevent excessive inflammation and edema.

In one embodiment, the tumor is located in the eye, nose, throat, or anypart of the neck and head, mucosa, skin, tongue, throat, eye, esophagus,thyroid, salivary or lacrimal glands, bladder, genitourinary system,nose, brain that can be reached through the natural body orifices orthrough an artery or a vein using laser tube delivery device with itsfiber optic for laser delivery to the tumor.

For example, as shown in FIG. 2, one laser fiber optic device 68 may beinserted through the conjunctiva so as to treat a tumor 62 located onthe sclera 58 of the eye 50 by emitting light pulses generated a lightsource 70. In FIG. 2, it can be seen that the eye 50 includes a cornea52, a lens capsule 54, a vitreous cavity 56, a sclera 58, and a retina60. In addition, as also shown in FIG. 2, another fiber optic device 72with light source 74 may be inserted into the vitreous cavity 56 of theeye 50 so as treat another tumor 66 located proximate to the retina 60of the eye 50. Alternatively, or in addition to the internally disposedfiber optic device 72, a light source 80 located outside of the eye 50may be used to deliver light pulses for treating the tumor 66. In FIG.2, it can be seen that the eye 50 contains another tumor 64 on theretina 60, which may be treated with the fiber optic device 72 or theexternal light source 80. Also, as shown in FIG. 2, a photoacousticreceiver 76 with cord 78 may be placed against the eyelid of the eye 50in order to record a photoacoustic/thermoacoustic response resultingfrom the thermal expansion of nanoparticles attached to the tumor (e.g.,attached to tumor 66 in the eye 50) that are heated by the laser lightpulses from a fiber optic device 72. As described above, thenanoparticles may be delivered to the tumor site prior to the heatingthereof by a tube attached to the fiber optic device 72.

As another example, as shown in FIG. 4, a laser fiber optic device 108may be inserted through the urethra so as to treat a tumor 106 locatedin the bladder 102 by emitting light pulses generated a light source110. As illustrated in FIG. 4, in addition to the bladder 102, it can beseen that the renal/urinary system illustrated therein further includesthe suprarenal glands 98, kidneys 100, and prostate 104.

As yet another example, as shown in FIG. 6, a laser fiber optic device140 may be inserted through the trachea 132 and bronchi 136 so as totreat a tumor 138 located in the lung 134 by emitting light pulsesgenerated a light source 142.

In another embodiment, polymeric antibody-coated nanoparticles orpolysaccharide or synthetic polymers conjugated with biomarkers andnanoparticles with cell penetrating peptides (CPP), activating CPP(ACPP), biotin, and/or streptavidin or antibody-coated nanoparticlesconjugated with (alpha)-cyclodextrin, (beta)-cyclodextrin,(gamma)-cyclodextrin, hydroxypropyl-b-cyclodextrin (bHPCD) areadministered to enhance a vaccination effect and are taken up by antigenpresenting cells.

In one embodiment, simultaneous administration of vaccine adjuvants withantibody-coated pluralities of nanoparticles with cell penetratingpeptides (CPP), activating CPP (ACPP), biotin, and/or streptavidin orantibody-coated nanoparticles conjugated with (alpha)-cyclodextrin,(beta)-cyclodextrin, (gamma)-cyclodextrin, hydroxypropyl-b-cyclodextrin(bHPCD) conjugated tumor lysates, VLPs, or purified antigen with orwithout adjuvant, interferon stimulating genes, toll-like receptors 7/8using thermosensitive polymers or without the use of thermal energy withantibody-coated chitosan or other slow release antibody-coatednanoparticles of biodegradable polymeric nanoparticles, lactic acid,glycolic acid, or combination of them. PLGA nanoparticles orpolycaprolactone, polyanhydrides, acrylamide, anhydride, or poroussilicon nanoparticles and/or microparticles, etc. stimulates immune cellresponse initially during the thermotherapy and afterwards keep immunecell, dendritic cells, T cells, activated natural killer (NK) cellsstimulation active for 3-6 months after the initial thermotherapytreatment or can be repeated as a vaccination every 6 months to killtumor cell recurrences locally or elsewhere in the body.

In another embodiment, the thermotherapy is associated with local,intra-arterial injection supplying an organ having the tumor,administration of immune stimulating agent conjugated withantibody-coated pluralities of nanoparticles or porous siliconnanoparticles and/or microparticles with cell penetrating peptides(CPP), activating CPP (ACPP), biotin, and/or streptavidin orantibody-coated nanoparticles conjugated with (alpha)-cyclodextrin,(beta)-cyclodextrin, (gamma)-cyclodextrin, hydroxypropyl-b-cyclodextrin(bHPCD) with tumor lysates, VLPs, or purified antigens with or withoutadjuvants, interferon stimulating genes, toll-like receptors 7/8 usingthermosensitive polymers or with or without the use of thermal energywith antibody-coated polymers, such as chitosan or other slow releaseantibody-coated nanoparticles of biodegradable polymeric nanoparticles,lactic acid, glycolic acid, PLGA nanoparticles, anhydride, etc. so as toexpose mostly the local invasive tumors, such as glioblastoma, arebathed with the antibody-coated nanoparticles rather than the entirebody, where the antibody coated nanoparticles or the immune stimulatorscould produce an adverse effect, such as in liver or kidney, etc.

In one embodiment, the local intra-arterial immune stimulatory therapyand thermotherapy is associated with culture grown immune cell therapy,where the number of CAR-T cell administration is kept far below 1/10 ofwhat would be needed for the whole body intravenous administration as isdone routinely, thus reducing the chance of an auto immune response.

In one embodiment, the venous drainage of an organ can be cannulated toremove blood containing the excessive cellular immune cells ornanoparticles, associated with checkpoint inhibitors or medicationscleansed from the cellular elements and nanoparticles and re-infused tothe patient's body to reduce or eliminate adverse effect of the systemicmedication as is done routinely.

In one embodiment, Phosphoinositides 3-Kinase are involved in cellsignaling pathways and play an important role in cellular growth,translation and metabolism. In general the Phosphoinositides 3-Kinaseare a part of PI4K/AkT/mTOR pathway which are involved in tumor growth.Their inhibition has been an important goal of cancer therapy. However,until now, the PI4K/Aky/mTOR pathway inhibitors and other isoforms ofthe PI3Ks with their subunits known as p110 alpha, beta, gamma and deltainhibitors such as wortmannin, Idelalisib, Alpelesib, Buperlasib, etc.are administered systemically and not selectively, locally,intra-arterially or intravenously along with pluralities of antibody ormonoclonal antibody-coated nanoparticles or antibody-coatednanoparticles conjugated with (alpha)-cyclodextrin, (beta)-cyclodextrin,(gamma)-cyclodextrin, hydroxypropyl-b-cyclodextrin (bHPCD) at far lowerdoses than used for systemic medication, while enhancing cellularpermeability with localized thermotherapy.

In one embodiment, similarly Phosphoinositides 3-Kinase or PI4Kinhibitors or Shep 2 inhibitors affect growth of certain tumors, such asintestinal, brain, lung, etc. cancers, but also affect proliferation ofmacrophages which interestingly support the proliferation of the tumorcells, this process of inhibition of macrophage proliferation can becountered with local administration of Phosphoinositides 3-Kinase orPI4K inhibitors, inhibitors or Shep 2 inhibitors by antibody-coatednanoparticles with cell penetrating peptides (CPP), activating CPP(ACPP), biotin, and/or streptavidin or antibody-coated nanoparticlesconjugated with (alpha)-cyclodextrin, (beta)-cyclodextrin,(gamma)-cyclodextrin, hydroxypropyl-b-cyclodextrin (bHPCD) areconjugated with viral-like particles (VLP), toxins, or lytic viruses notonly to damage the tumor cells but induce localized immune response,inflammation to attract the patient's lymphocytes, and killer cells andantibody-coated nanoparticles and slow release polymers of chitosans,polycaprolactone, lactic, and glycolic acid, anhydride or porous siliconnanoparticles and/or microparticles administration conjugated withimmune stimulators and checkpoint inhibitors, and immune stimulatorssuch as VLP or toxins enhancing innate humoral and other cellularresponse, such as natural killer cell T-lymphocytes, etc.

In one embodiment, pluralities of antibody or monoclonal antibody-coatednanoparticles or antibody-coated nanoparticles conjugated with(alpha)-cyclodextrin, (beta)-cyclodextrin, (gamma)-cyclodextrin,hydroxypropyl-b-cyclodextrin (bHPCD) are conjugated with thermosensitivenanoparticles conjugated with Phosphoinositides 3-Kinas inhibitors or incombination with AkT/mTOR inhibitors carrying immune stimulating agents,such as interferons, toll like receptors, toxins, or VLP or simultaneouscheckpoint inhibitors, such as PD-1, CTLA-4 or Jagged1, which can beinjected into the circulation intravenously or intra-arterially, and canbe released with the medication specifically at the site of themalignant tumors at a controlled temperature of 40-43 degrees C. orapplied locally or externally using laser light, focused ultrasound, ormicrowaves, RF, or an alternating magnetic field along with magneticnanoparticles, or magnetic luminescent porous silicon nanoparticlesand/or microparticles etc., and control of the energy by athermoacoustic imaging unit to treat the lesions by inhibiting thePI4K/AkT/mTOR Shp 2 pathway locally and enhance vascular and cellularpermeability with localized thermotherapy and simultaneously enhancesystemic humoral and cellular response to kill the tumors.

In one embodiment, the treatment of cancer, such as brain cancer (e.g.,glioblastoma) or hematological malignant cells, such as lymphoma orleukemia, or breast cancer of head and neck, eye, skin or mucosa cancersor lung or intestinal tract cancers, or genitourinary cancers in caseswhere the cancers have become therapy resistant to one medication, itrequires administration of a combination of medication that when appliedtogether, may be more beneficial to the patients, but also enhance eachother's side effects of medications in these cases. In one embodiment,pluralities of antibody or monoclonal antibody-coated nanoparticles orantibody coated nanoparticles conjugated with (alpha)-cyclodextrin,(beta)-cyclodextrin, (gamma)-cyclodextrin, hydroxypropyl-b-cyclodextrin(bHPCD) are conjugated with thermosensitive nanoparticles conjugatedwith Phosphoinositides 3-Kinas inhibitors, or in combination withAkT/mTOR inhibitors, such as everolimus, or Wnt inhibitors or Rhoinhibitors carrying immune stimulating agents, such as interferons,toll-like receptors, toxins, or VLP or simultaneous checkpointinhibitors, such as PD-1, CTLA-4 or Jaggedl can be injected into thecirculation intravenously or intra-arterially to be releasedspecifically the medication at the site of the malignant tumors at acontrolled temperature of 40-43 degrees C. or applied locally orexternally using laser light, focused ultrasound, or microwave, RF or analternating magnetic field along with magnetic nanoparticles, etc., andcontrol of the energy by a thermoacoustic imaging unit treat the lesionsby inhibiting the PI3K/AkT/mTOR or Shp 2 pathway locally and enhancevascular and cellular permeability with localized thermotherapy andsimultaneously enhance systemic humoral and cellular response to killthe tumors.

With respect to gene delivery, the inventive method may be used incancer therapy, but is not limited to such use; it will be appreciatedthat the inventive method may be used for gene delivery in general withantibody coated pluralities of nanoparticles with cell penetratingpeptide (CPP), activating CPP (ACPP), biotin, streptavidin. For example,the inventive method facilitates cellular gene uptake by current methodsthat lack a thermal energy component, such as electroporation, quantumdot delivery, etc. The controlled and precise application of thermalenergy enhances gene and medication transfer to any cell, whether thecell is a neoplastic cell, a pre-neoplastic cell, or a normal cell byusing CRISPR-cas9 mediated Homology-Independent Targeted Integration(HITI) or Homology Directed Repair (HDR).

The inventive method provides in vitro and in vivo precisionimmunotherapy to decrease or eradicate a malignant neoplasm at an earlystage of the disease. This method provides a vaccination effectperiodically to prevent at least the same kind of cancer or to treatrecurrences.

One embodiment is a method for evaluating treatment outcome in a patienthaving a genetic predisposition for a malignant neoplasm before clinicalmanifestation of the neoplasm can be seen radiographically. The methodpermits visualization of any tumor, whether located externally on apatient's body or located internally in the body, and as small as 2 mmin diameter, producing a biomarker, either a biomarker specific for thetumor or a general biomarker.

In general, a biomarker indicates a disease process. As subsequentlydescribed, a biomarker can be a protein, antigen, enzyme, hormone,carbohydrate, toxin, DNA, an organism such as bacteria, tumor cell,exosome, or indirectly an antibody, present in a liquid biopsy specimen.It can be produced by the plasma cells, against a tumor antigen, etc.

The method uses antibodies conjugated with nanoparticles with cellpenetrating peptides (CPP), activating CPP (ACPP), biotin, and/orstreptavidin, which include but are not limited to quantum dots, withthe conjugated form collectively termed functionalized nanoparticles,that are heated under specified conditions to produce a photoacoustic orthermoacoustic signal that is then then recorded and visualized tolocate the tumor to which the nanoparticles are attached. Nanoparticlesmay be used for qualitative and quantitative assessment of an analyte inthe blood or other tissue using photoacoustic/thermoacoustic technology,U.S. Pat. No. 8,554,296. As previously stated, as used herein, unlessspecifically stated otherwise, nanoparticles include but are not limitedto quantum dots.

Early stage small neoplastic cells produce biomarkers that are eitherspecific to the tumor cells or that represent the body's response to thetumor as an antibody. The biomarkers can be proteomic, genetic,epigenetic or glycomic biomolecules. These biomolecules can berecognized in the patient's tissue samples or in the blood or fluids.Their existence can be demonstrated thus far chemically using, e.g.,immunoassay or PCR methods. Quantitation of these biomarkers is alsoimportant to determine disease progression and prognosis of the disease.

Biomarkers for many diseases are found in the blood. As subsequentlydisclosed, biomarkers detected in a liquid biopsy sample are used togenerate antibodies against them using known methods in the art. Theanti-tumor antibodies are used to coat nanoparticles with cellpenetrating peptides (CPP), activating CPP (ACPP), biotin, and/orstreptavidin in the inventive method, where a lesion can be imagedregardless of the lesion size or location in the body. The method is notlimited to tumor detection and/or therapy. As only one example,detecting an antibody against anti-beta-amyloid protein present inAlzheimer's disease in a liquid biopsy specimen, the method renders theplaque visible with the nanoparticles and accessible to the inventivetreatment. As another example, the method can also be used to detectand/or treat inflammatory processes, etc.

The inventive method is applicable to any processes or diseases thatproduce a biomarker detectable in a liquid biopsy specimen. It isapplicable to a lesion including an abscess, an ulcer, a tumor eitherbenign or malignant, an ischemic area of a stroke and/or an area of thebrain affected by a stroke whether visible or microscopically.

Well over a thousand proteins are differentially expressed in humancancers and thus may serve as biomarkers. Such proteins play a role incancer-related processes such as angiogenesis, apoptosis, celldifferentiation, cell signaling, hematopoiesis, hormonal control, immunereactions, etc. Exemplary biomarkers include, but are not limited to,CEA for both malignant pleural effusion and peritoneal cancerdissemination; HER-2/neu for stage IV breast cancer; bladder tumorantigen for urothelial cell carcinoma; thyroglobulin for thyroid cancermetastasis; α-fetoprotein for hepatocellular carcinoma; PSA for prostatecancer; CA 125 for non-small cell lung cancer; CA 19.9 for pancreaticcancer; CA 15.3 for breast cancer; the combination of leptin, prolactin,osteopontin, and IGF-II for ovarian cancer; the combination of CD98,fascin, sPIgR, and 14-3-3 eta for lung cancer; troponin I for myocardialinfarction, and B-type natriuretic peptide for congestive heart failure.While the previous nine proteins are the only approved markers forcancer testing to date, they are but a small fraction of the totalnumber of available biomarkers, and their sensitivity and specific vary.

Other common biomarkers include the estrogen receptor/progesteronereceptor (ER/PR), HER-2/neu, and ESFR for breast cancer, andTIMP-1-associated with serum HER2-positive breast cancer; KRAS andUGT1A1 for colorectal cancer; HER-2/neu for gastric cancer, c-KIT, CD20antigen, CD30, and FIP1L1-PDGRF alpha, and PDGFR for GIST; PhiladelphiaChromosome (BCR/ABL)/PML/RAR alpha and TPMT/UGT1A1/ALK EGFR forleukemia/lymphoma; KRAS/EGFR for lung cancer, and BRAF and S100 formelanoma.

Other examples of biomarkers include tumor suppressors that are lost incancers, such as BRCA1, BRCA2; RNA such as mRNA, microRNA; proteinsfound in body fluids or tissue such as prostate specific antigen andCA-125; gene and protein based biomarkers; and nonspecific biomarkerssuch as glycosaminoglycans in body fluids; alkaline phosphatase andurinary hydroxyproline in skeletal involvement; hyaluronic acidexcretion and urinary hydroxyproline in bone disease, and combinationsthereof.

In malignancies, the biomarkers may be released into the circulationeither prior to or after the tumor has grown sufficiently to becomemetastatic. Small tumors (less than about 2 mm) seldom have any clinicalmanifestations, however even such small tumors can release chemicaland/or biomarkers into the circulation.

The existence of biomarkers in the circulation has been known, but hasnot met the threshold for locating tumor cells that could not be imagedradiographically or by ultrasound as long as the tumors wereasymptomatic. Available imaging methods such as x-ray, magneticresonance imaging (MRI), functional MRI, computed tomography (CT) scans,CT ultrasound, etc. may not permit visualization of lesions smaller thanabout 3 mm in diameter. This has been the case for most malignanttumors, or when a malignant tumor is created from a benign precursorlesion such as nevus, breast unspecific cyst or unspecific scar,prostate tumors along with benign prostate hypertrophy, or uterus cancerinside the uterus fibroma, melanoma inside a skin nevus or choroidalnevus under the retina in the eye or in a seborrheic keratosis, etc.Moreover, it is often difficult to follow a cancerous tumor which hasbeen irradiated but may still harbor malignant cells, and that can startgrowing with time and metastasize before it shows a local growth that isdetected by conventional imaging or other methods.

The diagnosis of a malignant tumor may be extremely difficult, even whena tumor is visible clinically or radiologically, e.g. a uterus fibromathat may have some malignant transformation. Moreover, a diagnosis alsoaffects the decision whether or not and also how to remove the tumor. Asone example, accessing the uterus through a small incision, and removingthe tumor piece by piece using an endoscope and a cutting probe, has afast post-operative recovery. Such a method is in contrast to completelyremoving the uterus with the tumor intact out of caution that the tumormay harbor neoplastic cells, but using a large incision withsignificantly higher operative risks and post-operative complicationprobabilities. Another, more problematic example, is the decision for awoman having genetic disposition to breast cancer without any physicalor radiological manifestation. The woman must endure the stress and fearnot knowing if or when she may develop breast cancer, and must considerprophylactic removal of both breasts. As another example, a personaldecision whether or not to undergo radiation therapy when a nevus isdiscovered under the retina, and biopsy results that often do notprovide definitive information because of the diversity of the cells inthe entire area of the tumor.

When the tumor site is unknown, locating a biomarker in the circulationmay be akin to finding a needle in a hay stack. For any particular tumoror cancer, not all biomarkers are even known. Similarly, finding a microDNA in the circulation may not provide an answer when the tumor iseither invisible or has already metastasized. An example of this occursin patients with uveal melanomas, having a mortality rate of about 50%,even if the tumors undergoes radiation, at the time the ophthalmologistdiscovers the tumor. This points to the fact that a malignant tumor canmetastasize very early, at times even when the size of the tumor isabout 2 mm in diameter which is equal to about one million cells. Ingeneral, these lesions do not have any symptoms.

The inventive method makes it possible to evaluate a patient withgenetic predisposition of a malignant neoplasm before its clinicalmanifestation can be seen radiographically.

In one embodiment, the presence of one or more biomarkers is evaluatedin any body fluid or organ. Exemplary bodily fluids include, but are notlimited to, urine, blood, cerebrospinal fluid (CSF), eye cavity fluid,tear film, sputum, fluid obtained from the trachea, bronchi, abdominalcavity, vagina, uterus etc. The biomarkers are analyzed in vitro bymethods known in the art, e.g., immunoassays including enzyme-linkedimmunoassay (ELISA), Western blots, fluorescence in situ hybridization(FISH), polymerase chain reaction (PCR), etc. The biomarkers are thenconjugated with functionalized antibody coated nanoparticles and/orquantum dots, as known in the art.

In one embodiment one obtains a liquid biopsy sample. Such a sample maybe obtained from, e.g., blood, urine, cerebrospinal fluid (CFS), aqueousor vitreous or abdominal cavity fluid, lymph node fluid, bladder fluid,milk duct fluid, sputum, gastric fluid, bile duct fluid, sinus fluid,etc. The patient may or may not have any clinical symptom. The patientmay or may not have history of a family disposition for tumors in and/orcancer of the breast, brain, lung, prostate, ovary, pancreas, etc., or agenetic abnormality leading to progression in diseases such as, e.g.,Alzheimer's, Parkinson's, post traumatic brain syndrome, brain tumor,other neurological disease, age related macular degeneration, aninfectious disease, an immune response, etc. The method evaluates thecomponents of the sample for cell free nucleic acid-based biomarkersincluding but not limited to microRNA and microDNA; protein-basedbiomarkers, extracellular vesicle (EV)-based biomarkers that arecontained within exosomes, extracellular vesicles, or microvesicles, andcirculating tumor cell (CTC)-based biomarkers. The method usesmethodologies such as next generation sequencing (NGS) or recombinantaffinity reagents fabricated into nanostructures such as carbonnanotubes, nanowires, quantum dots, or gold nanoshells, to enhance theirdetection with the use of, e.g., surface-enhanced Raman scattering(SERS), as known in the art.

For example, if a known tumor exists and there is a known biomarker forthe tumor, one may have or prepare an antibody against the tumor to beused in both imaging and therapy. Large tumors with symptoms can beimaged, but before the inventive method, there was a problem when abiomarker was present in a liquid biopsy specimen but the tumor wasinvisible, e.g., an early stage of a tumor, and there was no symptomaticor radiographic evidence of the tumor.

Detecting a tumor biomarker, typically a protein or a glycoprotein, in aliquid biopsy specimen is facilitated by the inventive method. Oncedetected, an antibody against that tumor biomarker can be prepared. Theantitumor biomarker antibody is used to locate the tumor. Antibodyproduction is a well-known method in the art, and it will be appreciatedthat the antibody against either or both of the tumor biomarker and thetumor cell may be recombinant, monoclonal, polyclonal, or an aptamer.The prepared antitumor cell antibodies are conjugated with nanoparticlesand administered to a patient, where they target the tumor cells and canbe detected and/or treated. Detection is by photoacoustic/thermoacousticimaging technology. Treatment is at least by one of thermal energy. Thephotoacoustic detection and thermal treatment is described herein.

In one embodiment, any specific tumor related biomarker may be used. Oneexample uses trastuzumab or herceptin, a recombinant monoclonalantibody, against the oncogene HER-2, previously mentioned, which is amember of the human epidermal growth factor receptor (HER/EGFR/ERBB)family. Other examples of known monoclonal antibodies or biologicsinclude, but are not limited to, rituximab, cetuximab, racotunomab,obinotuzumab, pertuzumab, belaniatumomab, bevacizumab, nivolumab,ofatumumab, botezomib, daratumumab, ipilumumab, pembrolizumab, anddaratumumab.

In one embodiment, in the absence of a specific biomarker, antibodiesagainst biomarkers that are shared by a number of the tumors may beused. Such biomarkers include glycosaminoglycan, which is specific for agroup of cancers such as bladder, gastrointestinal, glioblastoma, etc.Antibodies against such biomarkers are then conjugated withnanoparticles, termed functionalized nanoparticles. The term“functionalized” indicates nanoparticles that have been coated to renderthem soluble, biocompatible, and/or targeted by conjugating them with abiomolecule such as an antibody.

In one embodiment, the pluralities of nanoparticles may be one or moreof the following compounds or contain one or more of the followingcomponents: quantum dots, nanowires, nanotubes, nanoshells, nanocages,periodic mesoporous organosilica nanoparticles, perovskites,nanoparticles that are magnetic such as iron or iron oxide,paramagnetic, or nanoparticles that are non-magnetic such as gold,gold-silica, gold-iron, silica coated gold nanospheres and nanorods,copper sulfide, ferritic, quartz, graphene, carbon, zinc oxide,piezoelectric, porous silicon nano and microparticles or magneticluminescent porous silicon nanoparticles and/or microparticles, etc. Anyof these nanoparticles, alone or in combination, may be conjugated orotherwise associated with the biomarkers' antibodies, with cellpenetrating peptides (CPP), activating CPP (ACPP), biotin, and/orstreptavidin using methods known in the art.

In another embodiment, self-assembling bio/nano hybrid materialconsisting of two constituents at the nanometer or molecular levelcomposed of inorganic and organic compounds, having amphiphiliccharacteristics, i.e., hydrophilic and lipophilic components ormicelles, which may be radioactive (e.g., Cu⁶⁴)or radioactive (e.g.,tin) are prepared with biocompatible coatings with cell penetratingpeptides (CPP), activating CPP (ACPP), biotin, and/or streptavidin andadministered in the body for both therapy and imaging.

In one embodiment, the functionalized nanoparticles or magneticluminescent porous silicon nanoparticles and/or microparticles travel inthe body and attach to receptors of desired cells, e.g., tumors,Alzheimer's plaque, drusen of the retina, etc. These nanoparticles areimaged by applying external thermal energy and/or by applying anelectromagnetic radiation, microwaves, radiofrequency waves or areversible or alternating magnetic field. The thermal energy causes thenanoparticles to expand, producing an ultrasound wave in the tissueknown as photoacoustic or thermoacoustic sound. The ultrasound wave canbe detected by an ultrasonic receiver which is imaged in two to threedimensional formats as a tomogram. In another embodiment the plaques inAlzheimer's disease, and the drusen in age related macular degeneration,are rendered visible using silica coated nanoparticles<2 nm in diameteradministered with turmeric, glycosaminoglycan, amyloid antibody, orpercolan, etc. and are quantified. In another embodiment, thenanoparticles are conjugated with porous silicon nanoparticles and/ormicroparticles or magnetic luminescent porous SI nanoparticles andmicroparticles and/or thermosensitive polymers, such as chitosan,polylactic poly glycolic acid, acrylic derivatives, polycaprolactone,and are conjugated with are conjugated with antibodies, medications,sterols, antibiotics, antifungals, antibacterials, antiproliferativeagents, medications interfering with the normal cell signalingprocesses, with stimulatory or inhibitory action such as Wnt inhibitors(e.g., ivernectin) or Rho kinase inhibitors known as Rock inhibitors(e.g., Fasudil or Botox) or dyes, etc. that can be released from silicacoated gold nanoparticles when coated with thermosensitive polymers,e.g., chitosan coated nanoparticles heated to 40° C.-42° C., to treatvarious diseases including bacteria, fungi, parasites, plaque, drusen,inflammation, tumors, etc. In another embodiment, the plaques and drusencan be quantified by imaging using light, MRI,photoacoustic/thermoacoustic technology imaging, etc.

In another embodiment, the functionalized anti-biomarker-conjugatednanoparticle, ranges in size from 1 nm to 900 nm. In another embodiment,the functionalized biomarker ranges in size from 1 nm to 8 nm, chosen toenhance their elimination through the kidney for facilitated clearance.

In one embodiment, the nanoparticles are rendered magnetic by coatingwith a thin film of iron oxide prior to their conjugation withbiomarkers' antibodies.

In one embodiment, the nanoparticles are rendered more biocompatible bycoating with a compound, including but not limited to the following:(poly)ethylene glycol, cell penetrating peptide (CPP), activating CPP(ACPP), biotin, streptavidin, etc., as known in the art, prior to theirinjection in the body.

Thermal energy in the form of electromagnetic radiation, ultrasound, oran alternating magnetic field is applied, under the control of aphotoacoustic/thermoacoustic imaging system, to the organ suspected ofpotentially harboring an as yet invisible neoplasm. The thermal energyapplied increases preferentially the temperature of the exposednanoparticle, and creates (e.g. using a laser light) a photoacousticsound from the superficial lesions and or using focused ultrasound,microwave, RF, or an alternating magnetic field to produce athermoacoustic sound from deep in the tissue located lesions, and toimage or create a tomogram of the accumulated heatednanoparticles/tumor. This image or tomogram represents a suspectedneoplasm in that organ, and is compared to an image taken without thethermal application radiographically.

In one embodiment, one administers functionalized antibody-coatednanoparticles that, once attached to tumor cells, become visible with aphotoacoustic/thermoacoustic imaging unit that corroborates with animage obtained or not seen with other technology such as ultrasound,MRI, PET, CT scan, etc. In one embodiment, the images obtained withother instruments are either overlapped using a processor or are takensimultaneously during photoacoustic/thermoacoustic imaging. In oneembodiment, after administration of the antibody-coated nanoparticle,porous silicon nanoparticles and/or microparticles or magneticluminescent porous silicon nanoparticles and/or microparticles, periodicmesoporous organosilica nanoparticles an MRI image is overlapped withthe photoacoustic image and compared by a processor to verify thechanges in the imaged area.

In one embodiment, the nanoparticles are incorporated in liposomes orsolid lipid nanoparticles. In this embodiment, they may containmedications or a dye that, upon attainment of a specific tumortemperature, are released. The type of medication is not limited, andcan include anti-bacterial, anti-viral, anti-fungal, antineoplastic,anti-inflammatory such as acetyl cycline, anti-beta-amyloid protein,other antibodies, non-steroidal anti-inflammatory drugs, Rockinhibitors, Botox, Wnt inhibitors niclosamide, ivernectin, preventinginflammation or tumor growth, checkpoint inhibitors, anti-VEGF, immunestimulating agents, VLPs, anti-VEGF agents, propranolol,anti-aggregation agents, such as sterols, etc.

In another embodiment, antibody-coated nanoparticles or porous siliconnanoparticles and/or microparticles or magnetic luminescent poroussilicon nanoparticles and/or microparticles or antibody coatednanoparticles conjugated with (alpha)-cyclodextrin, (beta)-cyclodextrin,(gamma)-cyclodextrin, hydroxypropyl-b-cyclodextrin (bHPCD) andconjugated with thermosensitive polymers, such as chitosan, carrying anymedication including but not limited to sterol, squalamine, lanosterol,is administered to a patient having a neurologic pathology such asAlzheimer's disease, Parkinson's disease, or age related retinal drusen,etc. In this embodiment, administration is either intravenous or localin the cerebrospinal fluid or vitreous cavity, respectively, or atanother local site. After controllably increasing the temperature of thefunctionalized nanoparticle to between 40° C.-43° C. by increased energydelivery through a delivery source, under the control of thephotoacoustic imaging system and a processor, the temperature-sensitivecoating polymers such as chitosan melts and release medications specificto the pathology. For example, a medication to dissolve amyloid plaqueswould be administered to a patient with Alzheimer's disease; amedication to remove retinal drusen would be administered to a patientwith age related retinal disease, etc.

In one embodiment, the functionalized nanoparticle, e.g., a nanoshell,nanocage, porous silicon nanoparticles and/or microparticles or magneticluminescent porous silicon nanoparticles and/or microparticles, etc. iscombined with biodendrimers that are conjugated with biomarkers andmonoclonal antibodies and/or genes, e.g., siRNA, mRNA, RNAi, CRISPR toreduce the expression of checkpoint proteins by the tumor cells etc.,and for simultaneous visualization and therapy.

In another embodiment, after thermal imaging one increases thetemperature of the functionalized nanoparticles. This is achieved byincreased energy delivered by a thermal delivery source under thecontrol of the photoacoustic/thermoacoustic imaging system connected toa processor. The energy delivery unit increases the temperature of thefunctionalized nanoparticles to 41° C.-43° C. to melt thetemperature-sensitive coating polymers such as chitosan, liposomes orsolid lipid nanoparticles, and release anticancer medications, Rockinhibitors (e.g., fasudil, exoenzyme or Y27632, Botox etc.), Wntinhibitors (e.g., niclosamide, etc.) or inhibitory genes, siRNA, miRNA,RNAi, CRISPR to reduce the expression of checkpoint proteins by thetumor cells, etc., or checkpoint inhibitors, or introduce missing genes,or add any other genes for gene editing from the thermosensitive coatingof the nanoparticles along with a CRISPR complex to modify the geneticcomposition of the tumor cells, etc. by using CRISPR-cas9 mediatedHomology-Independent Targeted Integration (HITI) or Homology DirectedRepair (HDR). In another embodiment, the temperature of thefunctionalized nanoparticles is increased, by the thermal delivery unitvia a processor under the control of the photoacoustic/thermoacousticimaging unit to image the temperature and control it to 45° C.-47° C.,to 47° C., or to 50° C. to kill the suspected tumor to which theantibody-coated nanoparticles are attached and release tumor antigens inthe circulation to attract and enhance a cellular immune response to atumor.

In one embodiment, one synthetizes hybrid, very small (1 nm-8 nm) goldsilica nanoparticles that have a dual function, the nanoparticlesantibody coated for imaging, and having photovoltaic and magneticproperties, to release one or more gene(s) or medication(s) at certaintemperatures, creating a photoacoustic/thermoacoustic signal afterheating for imaging in the body or by laser or light stimulation in theeye for simultaneous imaging and therapy.

In one embodiment, using antibody coated quantum dots and light of aspecific wavelength that is absorbed by the quantum dot and emits lightof a different wavelength, one can render the moving tumor cells andextracellular vesicle visible attached to the quantum dots visible inthe retinal or choroidal vessels, or vessels and tumors of the skin, ortumors located beneath the skin and their feeding vessels, by lightabsorbed by the quantum dots circulating in the vessels, as is done influorescence angiography with appropriate filters and camera.

In another embodiment, a gold quantum dot in a mesoporous silica shellor cage is coated with an antibody or a biomarker with cell penetratingpeptides (CPP), activating CPP (ACPP), biotin, and/or streptavidin, orantibody coated nanoparticles conjugated with (alpha)-cyclodextrin,(beta)-cyclodextrin, (gamma)-cyclodextrin, hydroxypropyl-b-cyclodextrin(bHPCD) to any cell, e.g., neuronal or tumor cells, retinal drusen,Alzheimer plaques, etc. for delivering medication or gene modificationto an organ, e.g., retina or brain by using CRISPR-cas9 mediatedHomology-Independent Targeted Integration (HITI) or Homology DirectedRepair (HDR).

In another embodiment, the extent of plaque or drusen, as an indicatorof disease progression in the brain or eye, respectively, can beevaluated by conjugating nanoparticles with antibodies toglycosaminoglycan, heparin sulfate, glycosaminoglycan, and/or heparinsulfate proteoglycan, and injecting the composition into the circulationof the body or locally to adhere to plaques or drusen for diagnosis,quantitation, and/or therapy with antibodies and medication.

In another embodiment, the pluralities of antibody coated nanoparticleswith cell penetrating peptides (CPP), activating CPP (ACPP), biotin,and/or streptavidin or antibody-coated nanoparticles conjugated with(alpha)-cyclodextrin, (beta)-cyclodextrin, (gamma)-cyclodextrin,hydroxypropyl-b-cyclodextrin (bHPCD) are used for simultaneous imagingand thermotherapy of very small tumors. The nanoparticles are heated toa temperature ranging from 41° C.-43° C., releasing anti-cancermedication, such as Rock inhibitors, such as Botox, exoenzyme or Y27632,or Wnt inhibitors (e.g., niclosamide, ivermectin, Selamectin and alphalipoidic acid (ALA) precisely at the desired location, along withinhibitory siRNA, RNAi, CRISPR to reduce the expression of checkpointproteins by the tumor cells, etc., or modify a gene using theCRISPR/cas9 system or another CRISPR system mediatedHomology-Independent Targeted Integration (HITI) or Homology DirectedRepair (HDR), additionally releasing checkpoint inhibitors such asCTLA-4 or PD-1 or Jagged 1 along with tumoricidal vectors, etc.

In one embodiment, the pluralities of antibody coated nanoparticles withcell penetrating peptides (CPP), activating CPP (ACPP), biotin, and/orstreptavidin or antibody-coated nanoparticles conjugated with(alpha)-cyclodextrin, (beta)-cyclodextrin, (gamma)-cyclodextrin,hydroxypropyl-b-cyclodextrin (bHPCD) are rendered radioactive by coatingwith alpha or beta radiators that are antibody specific or nonspecificbiomarkers of the tumor. The nanoparticles can also be coated with heatsensitive polymers, including but not limited to chitosan, PEG, polyamino esters, etc.

In one embodiment, checkpoint inhibitors defined as immune systemcomponents that act as co-stimulatory or co-inhibitory molecules arereleased from the pluralities of antibody coated nanoparticles fromthermosensitive coating of the nanoparticles at temperature of 41 to 43degrees C. along with poisons such as bee or snake venom, ant, sodiumformate, or other toxic agents that damage tumor cell membranes, orgenes that inhibit tumor growth, siRNA, siDNA, mi RNA, mDNA along withthe CRISPR/cas 9 complex using CRISPR-cas9 mediated Homology-IndependentTargeted Integration (HITI) or Homology Directed Repair (HDR) orvariations of these may be used.

In one embodiment, the pluralities of antibody coated nanoparticles withcell penetrating peptides (CPP), activating CPP (ACPP), biotin, and/orstreptavidin or antibody coated nanoparticles conjugated with(alpha)-cyclodextrin, (beta)-cyclodextrin, (gamma)-cyclodextrin,hydroxypropyl-b-cyclodextrin (bHPCD) are coated with a specific or anonspecific biomarker such as glycosaminoglycan and injected into thecirculation, into a body fluid such as the lymphatic system orcerebrospinal fluid (CSF), or inside a body cavity. Examples ofinjection sites include, but are not limited to, eye, sinuses, abdominalcavity, bladder, uterus, etc. The nanoparticles with cell penetratingpeptides (CPP), activating CPP (ACPP), biotin, and/or streptavidin mayalso be injected into the breast ducts, e.g., through the nipple, insidethe brain, into the prostate or other organ, or may even be appliedtopically. The injected nanoparticles circulate and seek cells bearing areceptor to their antibody, or perhaps cells with specific receptors orbiomolecules, and readily attach within minutes or hours.

In one embodiment, specific or non-specific biomarkers' antibodies areconjugated with nanoparticles with cell penetrating peptides (CPP),activating CPP (ACPP), biotin, and/or streptavidin or antibody coatednanoparticles conjugated with (alpha)-cyclodextrin, (beta)-cyclodextrin,(gamma)-cyclodextrin, hydroxypropyl-b-cyclodextrin (bHPCD), and injectedeither into circulation or locally into a body cavity. The nanoparticlestravel and seek cells bearing specific receptors or biomolecules, andattach within a few hours. The patient's body or organ is then scanned,with the thermal energy producing radiation or an alternating orreversible magnetic field, microwave radiation, a laser, radiofrequency(RF) waves or focused ultrasound to heat the nanoparticles. Usingphotoacoustic/thermoacoustic technology, the sound wave generated by thethermal expansion of the nanoparticle induced by absorption of thethermal energy is recorded. The sound wave signals may originate fromany part of the body, or from a specific organ.

In one embodiment, an alternating magnetic field produces heat inmagnetic nanoparticles as a result of rapid circular or semicircularmotion of the magnetic or paramagnetic nanoparticles heating them. Thepatient's body is scanned within the reversible magnetic field, and thephotoacoustic sound is recorded as a temperature profile of the site ofthe nanoparticle/cell membrane imaged and location of the lesion isverified.

In another embodiment, other sources of thermal energy are used. Suchsources include, but are not limited to, electromagnetic radiation,visible light, invisible light, infrared radiation, microwaves, orradiofrequency waves, focused ultrasound (FUS), etc. The nanoparticleswith cell penetrating peptides (CPP), activating CPP (ACPP), biotin,and/or streptavidin are heated from body temperature of 37° C. to 40° C.or 43° C., or if needed to 45° C. At the desired temperature, e.g., 41°C.-43° C., the heat sensitive coating of the nanoparticle melts,releasing its cargo of, e.g., medication, gene, etc., thus facilitatingor enhancing passage of these compounds through the membrane of theneoplastic cells and by using CRISPR-cas9 mediated Homology-IndependentTargeted Integration (HITI) or Homology Directed Repair (HDR).

In another embodiment, use of a photoacoustic technology unit controlsthe thermal delivery unit and the thermal energy delivered to thenanoparticles to maintain or reach a predetermined temperature for adesired time.

In one embodiment, the temperatures rise of the nanoparticles expandsthem, producing a photoacoustic or thermoacoustic sound wave. This soundwave is recorded by one or multiple ultrasonic receivers located on thepatient's skin. The signal can be obtained from any part of the body, orfrom a specific organ, since the signal travels through the body as awave. The signal or sound pulse is converted to an electric pulse in thereceiver, then using a processor, is amplified and imaged on a monitor.A processor produces a two- or three-dimension image of the lesion,localizing the location of the sound and indicating the size of a lesionand its temperature by the amplitude of the sound pulse.

In one embodiment, photoacoustic imaging is used for a very early stagediagnosis of cancerous lesion that are less than 2 mm in diameter, whichare radiographically invisible without knowing their exact location inthe body.

In one embodiment using photoacoustic technology and a specific ornon-specific tumor biomarker, a very small lesion (<2 mm in diameter) isimaged in the body when the tumor has not caused any clinical symptom.The inventive method thus is used to differentiate a malignant lesionfrom a benign lesion, even if the cancerous lesion is inside a beginlesion. It is noteworthy that biopsy of these very small tumors, evenwhen the lesion is visible, e.g., on skin or under the retina, may notyield malignant cells if the biopsy is performed on a part of the lesionthat contains benign cells. With tumors in the brain, it is most oftenthe case that the tumors will not be noted absent a neurologicalsymptom.

In one embodiment, the inventive method is used with specific biomarkersof a tumor such as breast cancer, prostate cancer, glioma, pancreaticmalignancies, along with nonspecific biomarkers. The location and sizeof a malignant tumor in any organ is imaged in a patient with a geneticpropensity to develop a tumor. The thermal energy may also be applied,if desired, to treat the lesion simultaneously with providing thephotoacoustic effect. Subsequent evaluation of the level of thesebiomarkers in the blood indicate if the lesion was damaged or eliminatedby the thermal energy increasing the biomarkers in the blood, includinguse of medicaments/dye released from the thermosensitive nanoparticlecoating with cell penetrating peptides (CPP), activating CPP (ACPP),biotin, streptavidin or antibody coated nanoparticles conjugated with(alpha)-cyclodextrin, (beta)-cyclodextrin, (gamma)-cyclodextrin,hydroxypropyl-b-cyclodextrin (bHPCD) and/or other treatment agentsdelivered by the method as cargo in the nanoparticles.

In one embodiment, a combination of biomarkers can be used in an earlystage. For example, specific or nonspecific bio-markers such asglycosaminoglycans can be used in imaging a malignant lesion usingantibody-coated nanoparticles to photoacoustically image the presence ofa very small early stage tumor anywhere in the body.

In another embodiment, the inventive method is employed to determineresidual tumor cells that may have left at the site of a tumor resectionor elsewhere in the body, and to treat or eliminate the residual tumorcells.

In another embodiment, the functionalized nanoparticles are conjugatedwith one of the recombinant, monoclonal, or polyclonal antibodies oraptamers known in the art and administered along with either one or moretoxin(s) or antibodies, along with a medication that is provided at amuch lower dose systemically to kill the already compromised tumorcells. Monoclonal antibodies that may be used include, but are notlimited to, those shown in Table 1, e.g., rituximab, obinuzumab,oftumumab, etc.

TABLE 1 Drug Trade name Type Source Target Use 3F8 mab mouse GD2neuroblastoma 8H9 mab mouse B7-H3 neuroblastoma, sarcoma, metastaticbrain cancers Abagovomab mab mouse CA-125 ovarian cancer (imitation)Abciximab ReoPro Fab chimeric CD41 (integrin platelet alpha-IIb)aggregation inhibitor Abituzumab mab humanized CD51 cancer Abrilumab mabhuman integrin α4β7 inflammatory bowel disease, ulcerative colitis,Crohn's disease Actoxumab mab human Clostridium Clostridium difficiledifficile infection Adalimumab Humira mab human TNF-α Rheumatoidarthritis, Crohn's Disease, Plaque Psoriasis, Psoriatic Arthritis,Ankylosing Spondylitis, Juvenile Idiopathic Arthritis, Hemolytic diseaseof the newborn Adecatumumab mab human EpCAM prostate and breast cancerAducanumab mab human beta-amyloid Alzheimer's disease Afelimomab F(ab′)₂mouse TNF-α sepsis Afutuzumab mab humanized CD20 lymphoma AlacizumabF(ab′)₂ humanized VEGFR2 cancer pegol ALD518 ? humanized IL-6 rheumatoidarthritis Alemtuzumab Campath, mab humanized CD52 Multiple sclerosisMabCampath Alirocumab mab human NARP-1 hypercholeterolemia AltumomabHybri-ceaker mab mouse CEA colorectal cancer pentetate (diagnosis)Amatuximab mab chimeric mesothelin cancer Anatumomab Fab mouse TAG-72non-small cell lung mafenatox carcinoma Anetumab mab human MSLN cancerravtansine Anifrolumab mab human interferon α/β systemic lupus receptorerythematosus Anrukinzumab mab humanized IL-13 ? (=IMA-638) Apolizumabmab humanized HLA-DR? hematological cancers Arcitumomab CEA-Scan Fab′mouse CEA gastrointestinal cancers (diagnosis) Ascrinvacumab mab humanactivin receptor- cancer like kinase 1 Aselizumab mab humanizedL-selectin severely injured (CD62L) patients Atezolizumab mab humanizedCD274 cancer Atinumab mab human RTN4 ? Atlizumab Actemra, mab humanizedIL-6 receptor rheumatoid arthritis (=tocilizumab) RoActemra Atorolimumabmab human Rhesus factor hemolytic disease of the newborn[citationneeded] Bapineuzumab mab humanized beta amyloid Alzheimer's diseaseBasiliximab Simulect mab chimeric CD25 (α chain of prevention of organIL-2 receptor) transplant rejections Bavituximab mab chimericphosphatidylserine cancer, viral infections Bectumomab LymphoScan Fab′mouse CD22 non-Hodgkin's lymphoma (detection) Begelomab mab mouse DPP4 ?Belimumab Benlysta, mab human BAFF non-Hodgkin LymphoStat-B lymphomaetc. Benralizumab mab humanized CD125 asthma Bertilimumab mab humanCCL11 (eotaxin-1) severe allergic disorders Besilesomab Scintimun mabmouse CEA-related inflammatory antigen lesions and metastases(detection) Bevacizumab Avastin mab humanized VEGF-A metastatic cancer,retinopathy of prematurity Bezlotoxumab mab human ClostridiumClostridium difficile difficile infection Biciromab FibriScint Fab′mouse fibrin II, beta thromboembolism chain (diagnosis) Bimagrumab mabhuman ACVR2B myostatin inhibitor Bimekizumab mab humanized IL17A andIL17F ? Bivatuzumab mab humanized CD44 v6 squamous cell mertansinecarcinoma Blinatumomab BiTE mouse CD19 cancer Blosozumab mab humanizedSOST osteoporosis Bococizumab mab humanized neural apoptosis-dyslipidemia regulated proteinase 1 Brentuximab mab chimeric CD30(TNFRSF8) hematologic vedotin cancers Briakinumab mab human IL-12, IL-23psoriasis, rheumatoid arthritis, inflammatory bowel diseases, multiplesclerosis Brodalumab mab human IL-17 inflammatory diseases Brolucizumabmab humanized VEGFA ? Brontictuzumab mab Notch 1 cancer CanakinumabIlaris mab human IL-1? rheumatoid arthritis Cantuzumab mab humanizedmucin CanAg colorectal cancer mertansine etc. Cantuzumab mab humanizedMUC1 cancers ravtansine Caplacizumab mab humanized VWF thromboticthrombocytopenic purpura, thrombosis Capromab Prostascint mab mouseprostatic prostate cancer pendetide carcinoma cells (detection) Carlumabmab human MCP-1 oncology/immune indications Catumaxomab Removab 3functrat/mouse hybrid EpCAM, CD3 ovarian cancer, malignant ascites, gastriccancer cBR96- mab humanized Lewis-Y antigen cancer doxorubicinimmunoconjugate Cedelizumab mab humanized CD4 prevention of organtransplant rejections, treatment of autoimmune diseases CertolizumabCimzia Fab′ humanized TNF-α Crohn's disease pegol Cetuximab Erbitux mabchimeric EGFR metastatic colorectal cancer and head and neck cancerCh.14.18 mab chimeric ??? neuroblastoma Citatuzumab Fab humanized EpCAMovarian cancer and bogatox other solid tumors Cixutumumab mab humanIGF-1 receptor solid tumors Clazakizumab mab humanized Oryctolagusrheumatoid arthritis cuniculus Clenoliximab mab chimeric CD4 rheumatoidarthritis Clivatuzumab hPAM4-Cide mab humanized MUC1 pancreatic cancertetraxetan Codrituzumab mab humanized glypican 3 cancer Coltuximab mabchimeric CD19 cancer ravtansine Conatumumab mab human TRAIL-R2 cancerConcizumab mab humanized TFPI bleeding Crenezumab mab humanized1-40-β-amyloid Alzheimer's disease CR6261 mab human Influenza Ainfectious hemagglutinin disease/influenza A Dacetuzumab mab humanizedCD40 hematologic cancers Daclizumab Zenapax mab humanized CD25 (α chainof prevention of organ IL-2 receptor) transplant rejections Dalotuzumabmab humanized insulin-like cancer etc. growth factor I receptorDapirolizumab mab humanized CD40 ligand ? pegol Daratumuma mab humanCD38 (cyclic ADP cancer ribose hydrolase) Dectrekumab mab human IL-13 ?Demcizumab mab humanized DLL4 cancer Denintuzumab mab humanized CD19cancer mafodotin Denosumab Prolia mab human RANKL osteoporosis, bonemetastases etc. Derlotuximab mab chimeric histone complex recurrentbiotin glioblastoma multiforme Detumomab mab mouse B-lymphoma celllymphoma Dinutuximab mab chimeric ganglioside GD2 neuroblastomaDiridavumab mab human hemagglutinin influenza A Dorlimomab F(ab′)₂ mouse? ? aritox Drozitumab mab human DR5 cancer etc. Duligotumab mab humanHER3 ? Dupilumab mab human IL4 atopic diseases Durvalumab mab humanCD274 cancer Dusigitumab mab human ILGF2 cancer Ecromeximab mab chimericGD3 ganglioside malignant melanoma Eculizumab Soliris mab humanized C5paroxysmal nocturnal hemoglobinuria Edobacomab mab mouse endotoxinsepsis caused by Gram-negative bacteria Edrecolomab Panorex mab mouseEpCAM colorectal carcinoma Efalizumab Raptiva mab humanized LFA-1(CD11a) psoriasis (blocks T- cell migration) Efungumab Mycograb scFvhuman Hsp90 invasive Candida infection Eldelumab mab human interferonCrohn's disease, gamma-induced ulcerative colitis protein Elgemtumab mabhuman ERBB3 cancer Elotuzumab mab humanized SLAMF7 multiple myelomaElsilimomab mab mouse IL-6 ? Emactuzumab mab humanized CSF1R cancerEmibetuzumab mab humanized HHGFR cancer Enavatuzumab mab humanized TWEAKreceptor cancer etc. Enfortumab mab human AGS-22M6 cancer expressingvedotin Nectin-4 Enlimomab mab mouse ICAM-1 (CD54) ? pegol Enoblituzumabmab humanized B7-H3 cancer Enokizumab mab humanized IL9 asthmaEnoticumab mab human DLL4 ? Ensituximab mab chimeric 5AC cancerEpitumomab mab mouse episialin ? cituxetan Epratuzumab mab humanizedCD22 cancer, SLE Erlizumab F(ab′)₂ humanized ITGB2 (CD18) heart attack,stroke, traumatic shock Ertumaxomab Rexomun 3funct rat/mouse hybridHER2/neu, CD3 breast cancer etc. Etaracizumab Abegrin mab humanizedintegrin α_(v) β₃ melanoma, prostate cancer, ovarian cancer etc.Etrolizumab mab humanized integrin α₇ B₇ inflammatory bowel diseaseEvinacumab mab human angiopoietin 3 dyslipidemia Evolocumab mab humanPCSK9 hypercholesterolemia Exbivirumab^([) mab human hepatitis Bhepatitis B surface antigen Fanolesomab NeutroSpec mab mouse CD15appendicitis (diagnosis) Faralimomab mab mouse interferon ? receptorFarletuzumab mab humanized folate receptor 1 ovarian cancer Fasinumabmab human HNGF acute sciatic pain FBTA05 Lymphomun 3funct rat/mousehybrid CD20 chronic lymphocytic leukaemia Felvizumab mab humanizedrespiratory respiratory syncytial virus syncytial virus infectionFezakinumab mab human IL-22 rheumatoid arthritis, psoriasis Ficlatuzumabmab humanized HGF cancer etc. Figitumumab mab human IGF-1 receptoradrenocortical carcinoma, non- small cell lung carcinoma etc. Firivumabmab human influenza A virus ? hemagglutinin Flanvotumab mab humanTYRP1(glycoprotein melanoma 75) Fletikumab mab human IL 20 rheumatoidarthritis Fontolizumab HuZAF mab humanized IFN-γ Crohn's disease etc.Foralumab mab human CD3 epsilon ? Foravirumab mab human rabies virusrabies (prophylaxis) glycoprotein Fresolimumab mab human TGF-βidiopathic pulmonary fibrosis, focal segmental glomerulosclerosis,cancer Fulranumab mab human NGF pain Futuximab mab chimeric EGFR ?Galiximab mab chimeric CD80 B-cell lymphoma Ganitumab mab human IGF-Icancer Gantenerumab mab human beta amyloid Alzheimer's diseaseGavilimomab mab mouse CD147 (basigin) graft versus host diseaseGemtuzumab Mylotarg mab humanized CD33 acute myelogenous ozogamicinleukemia Gevokizumab mab humanized IL-1β diabetes etc. GirentuximabRencarex mab chimeric carbonic clear cell renal cell anhydrase 9carcinoma^([81]) (CA-IX) Glembatumumab mab human GPNMB melanoma, breastvedotin cancer Golimumab Simponi mab human TNF-α rheumatoid arthritis,psoriatic arthritis, ankylosing spondylitis Gomiliximab mab chimericCD23 (IgE allergic asthma receptor) Guselkumab mab human IL23 psoriasisIbalizumab mab humanized CD4 HIV infection Ibritumomab Zevalin mab mouseCD20 non-Hodgkin's tiuxetan lymphoma Icrucumab mab human VEGFR-1 canceretc. Idarucizumab mab humanized dabigatran reversal of anticoagulanteffects of dabigatran Igovomab Indimacis- F(ab′)₂ mouse CA-125 ovariancancer 125 (diagnosis) IMAB362 mab human CLDN18.2 gastrointestinaladenocarcinomas and pancreatic tumor Imalumab mab human MIF cancerImciromab Myoscint mab mouse cardiac myosin cardiac imaging Imgatuzumabmab humanized EGFR cancer Inclacumab mab human selectin P ? Indatuximabmab chimeric SDC1 cancer ravtansine Indusatumab mab human GUCY2C cancervedotin Infliximab Remicade mab chimeric TNF-α rheumatoid arthritis,ankylosing spondylitis, psoriatic arthritis, psoriasis, Crohn's disease,ulcerative colitis Intetumumab mab human CD51 solid tumors (prostatecancer, melanoma) Inolimomab mab mouse CD25 (α chain of graft versushost IL-2 receptor) disease Inotuzumab mab humanized CD22 cancerozogamicin Ipilimumab Yervoy mab human CD152 melanoma Iratumumab mabhuman CD30 (TNFRSF8) Hodgkin's lymphoma Isatuximab mab chimeric CD38cancer Itolizumab mab humanized CD6 ? Ixekizumab mab humanized IL 17Aautoimmune diseases Keliximab mab chimeric CD4 chronic asthmaLabetuzumab CEA-Cide mab humanized CEA colorectal cancer Lambrolizumabmab humanized PDCD1 antineoplastic agent Lampalizumab mab humanized CFD? Lebrikizumab mab humanized IL-13 asthma Lemalesomab mab mouse NCA-90diagnostic agent (granulocyte antigen) Lenzilumab mab human CSF2 ?Lerdelimumab mab human TGF beta 2 reduction of scarring after glaucomasurgery Lexatumumab mab human TRAIL-R2 cancer Libivirumab mab humanhepatitis B hepatitis B surface antigen Lifastuzumab mab humanizedphosphate- cancer vedotin sodium co- transporter Ligelizumab mabhumanized IGHE severe asthma and chronic spontaneous urticaria Lilotomabmab mouse CD37 cancer satetraxetan Lintuzumab mab humanized CD33 cancerLirilumab mab human KIR2D ? Lodelcizumab mab humanized PCSK9hypercholesterolemia Lokivetmab mab veterinary Canis lupus ? familiarisIL31 Lorvotuzumab mab humanized CD56 cancer mertansine Lucatumumab mabhuman CD40 multiple myeloma, non-Hodgkin's lymphoma, Hodgkin's lymphomaLulizumab pegol mab humanized CD28 autoimmune diseases Lumiliximab mabchimeric CD23 (IgE chronic lymphocytic receptor) leukemia Lumretuzumabmab humanized ERBB3 cancer Mapatumumab mab human TRAIL-R1 cancerMargetuximab mab humanized ch4D5 cancer Maslimomab ? mouse T-cellreceptor ? Mavrilimumab mab human GMCSF receptor rheumatoid arthritisα-chain Matuzumab mab humanized EGFR colorectal, lung and stomach cancerMepolizumab Bosatria mab humanized IL-5 asthma and white blood celldiseases Metelimumab mab human TGF beta 1 systemic sclerodermaMilatuzumab mab humanized CD74 multiple myeloma and other hematologicalmalignancies Minretumomab mab mouse TAG-72 tumor detection (andtherapy?) Mirvetuximab mab chimeric folate receptor cancer soravtansinealpha Mitumomab mab mouse GD3 ganglioside small cell lung carcinomaMogamulizumab mab humanized CCR4 cancer Morolimumab mab human Rhesusfactor ? Motavizumab Numax mab humanized respiratory respiratorysyncytial virus syncytial virus (prevention) Moxetumomab mab mouse CD22cancer pasudotox Muromonab- Orthoclone mab mouse CD3 prevention of organCD3 OKT3 transplant rejections Nacolomab Fab mouse C242 antigencolorectal cancer tafenatox Namilumab mab human CSF2 ? Naptumomab Fabmouse 5T4 non-small cell lung estafenatox carcinoma, renal cellcarcinoma Narnatumab^([) mab human RON cancer Natalizumab Tysabri mabhumanized integrin α₄ multiple sclerosis, Crohn's disease Nebacumab mabhuman endotoxin sepsis Necitumumab mab human EGFR non-small cell lungcarcinoma Nemolizumab mab humanized IL31RA ? Nerelimomab mab mouse TNF-α? Nesvacumab mab human angiopoietin 2 cancer Nimotuzumab Theracim, mabhumanized EGFR squamous cell Theraloc carcinoma, head and neck cancer,nasopharyngeal cancer, glioma Nivolumab Opdivo mab human PD-1 cancerNofetumomab Verluma Fab mouse ? cancer (diagnosis) merpentanObiltoxaximab mab chimeric Bacillus anthracis Bacillus anthracis anthraxspores Obinutuzumab Gazyva mab humanized CD20 Chronic lymphatic leukemiaOcaratuzumab mab humanized CD20 cancer Ocrelizumab mab humanized CD20rheumatoid arthritis, lupus erythematosus etc. Odulimomab mab mouseLFA-1 (CD11a) prevention of organ transplant rejections, immunologicaldiseases Ofatumumab Arzerra mab human CD20 chronic lymphocytic leukemiaetc. Olaratumab mab human PDGF-R α cancer Olokizumab mab humanized IL6 ?Omalizumab Xolair mab humanized IgE Fc region allergic asthmaOnartuzumab mab humanized human scatter cancer factor receptor kinaseOntuxizumab mab chimeric/humanized TEM1 cancer Opicinumab mab humanLINGO-1 multiple sclerosis Oportuzumab scFv humanized EpCAM cancermonatox Oregovomab OvaRex mab mouse CA-125 ovarian cancer Orticumab mabhuman oxLDL ? Otelixizumab mab chimeric/humanized CD3 diabetes mellitustype 1 Otlertuzumab mab humanized CD37 cancer Oxelumab mab human OX-40asthma Ozanezumab mab humanized NOGO-A ALS and multiple sclerosisOzoralizumab mab humanized TNF-α inflammation Pagibaximab mab chimericlipoteichoic acid sepsis (Staphylococcus) Palivizumab Synagis, mabhumanized F protein of respiratory Abbosynagis respiratory syncytialvirus syncytial virus (prevention) Panitumumab Vectibix mab human EGFRcolorectal cancer Pankomab mab humanized tumor specific ovarian cancerglycosylation of MUC1 Panobacumab mab human Pseudomonas Pseudomonasaeruginosa aeruginosa infection Parsatuzumab mab human EGFL7 cancerPascolizumab mab humanized IL-4 asthma Pasotuxizumab mabchimeric/humanized folate hydrolase cancer Pateclizumab mab humanizedLTA TNF Patritumab mab human HER3 cancer Pembrolizumab mab humanizedPDCD1 cancer etc. Pemtumomab Theragyn ? mouse MUC1 cancer Perakizumabmab humanized IL 17A arthritis Pertuzumab Omnitarg mab humanizedHER2/neu cancer Pexelizumab scFv humanized C5 reduction of side effectsof cardiac surgery Pidilizumab mab humanized PD-1 cancer and infectiousdiseases Pinatuzumab mab humanized CD22 cancer vedotin Pintumomab mabmouse adenocarcinoma adenocarcinoma antigen (imaging) Placulumab mabhuman human TNF ? Polatuzumab mab humanized CD79B cancer vedotinPonezumab^([120]) mab humanized human beta- Alzheimer's disease amyloidPriliximab mab chimeric CD4 Crohn's disease, multiple sclerosisPritoxaximab mab chimeric E. coli shiga toxin ? type-1 Pritumumab mabhuman vimentin brain cancer PRO 140 ? humanized CCR5 HIV infectionOuilizumab mab humanized IGHE asthma Tetulomab mab humanized CD37cancer^([121]) Racotumomab mab mouse N- cancer glycolylneuraminic acidRadretumab mab human fibronectin extra cancer domain-B Rafivirumab mabhuman rabies virus rabies (prophylaxis) glycoprotein Ralpancizumab^([)mab humanized neural apoptosis- dyslipidemia regulated proteinase 1Ramucirumab Cyramza mab human VEGFR2 solid tumors Ranibizumab LucentisFab humanized VEGF-A macular degeneration (wet form) Raxibacumab mabhuman anthrax toxin, anthrax protective antigen (prophylaxis andtreatment) Refanezumab mab humanized myelin-associated recovery of motorglycoprotein function after stroke Regavirumab mab human cytomegaloviruscytomegalovirus glycoprotein B infection Reslizumab mab humanized IL-5inflammations of the airways, skin and gastrointestinal tractRilotumumab mab human HGF solid tumors Rinucumab mab humanplatelet-derived neovascular age- growth factor related macular receptorbeta degeneration Rituximab MabThera, mab chimeric CD20 lymphomas,Rituxan leukemias, some autoimmune disorders Robatumumab mab human IGF-1receptor cancer Roledumab mab human RHD ? Romosozumab mab humanizedsclerostin osteoporosis Rontalizumab mab humanized IFN-α systemic lupuserythematosus Rovelizumab LeukArrest mab humanized CD11, CD18haemorrhagic shock etc. Ruplizumab Antova mab humanized CD154 (CD40L)rheumatic diseases Sacituzumab mab humanized tumor-associated cancergovitecan calcium signal transducer 2 Samalizumab mab humanized CD200cancer Sarilumab mab human IL6 rheumatoid arthritis, ankylosingspondylitis Satumomab mab mouse TAG-72 cancer (diagnosis) pendetideSecukinumab mab human IL 17A uveitis, rheumatoid arthritis psoriasisSeribantumab mab human ERBB3 cancer Setoxaximab mab chimeric E. colishiga toxin ? type-2 Sevirumab ? human cytomegalovirus cytomegalovirusinfection Sibrotuzumab mab humanized FAP cancer SGN-CD19A mab humanizedCD19 acute lymphoblastic leukemia and B-cell non-Hodgkin lymphomaSGN-CD33A mab humanized CD33 Acute myeloid leukemia Sifalimumab mabhumanized IFN-α SLE, dermatomyositis, polymyositis Siltuximab mabchimeric IL-6 cancer Simtuzumab mab humanized LOXL2 fibrosis Siplizumabmab humanized CD2 psoriasis, graft- versus-host disease (prevention)Sirukumab mab human IL-6 rheumatoid arthritis Sofituzumab mab humanizedCA 125 ovarian cancer vedotin Solanezumab mab humanized beta amyloidAlzheimer's disease Solitomab mab mouse EpCAM ? Sonepcizumab ? humanizedsphingosine-1- choroidal and phosphate retinal neovascularizationSontuzumab mab humanized episialin ? Stamulumab mab human myostatinmuscular dystrophy Sulesomab LeukoScan Fab' mouse NCA-90 osteomyelitis(granulocyte (imaging) antigen) Suvizumab mab humanized HIV-1 viralinfections Tabalumab mab human BAFF B-cell cancers Tacatuzumab AFP-Cidemab humanized alpha-fetoprotein cancer tetraxetan Tadocizumab Fabhumanized integrin α_(IIb)β₃ percutaneous coronary interventionTalizumab mab humanized IgE allergic reaction Tanezumab mab humanizedNGF pain Taplitumomab mab mouse CD19 cancer[citation needed] paptoxTarextumab mab human Notch receptor cancer Tefibazumab Aurexis mabhumanized clumping factor A Staphylococcus aureus infection TelimomabFab mouse ? ? aritox Tenatumomab mab mouse tenascin C cancer Teneliximabmab chimeric CD40 ? Teplizumab mab humanized CD3 diabetes mellitus type1 Teprotumumab mab human CD221 hematologic tumors Tesidolumab mab humanC5 ? TGN1412 ? humanized CD28 chronic lymphocytic leukemia, rheumatoidarthritis Ticilimumab mab human CTLA-4 cancer (=tremelimumab)Tildrakizumab mab humanized IL23 immunologically mediated inflammatorydisorders Tigatuzumab mab humanized TRAIL-R2 cancer TNX-650 ? humanizedIL-13 Hodgkin's lymphoma Tocilizumab Actemra, mab humanized IL-6receptor rheumatoid arthritis (=atlizumab) RoActemra Toralizumab mabhumanized CD154 (CD40L) rheumatoid arthritis, lupus nephritis etc.Tosatoxumab mab human Staphylococcus ? aureus Tositumomab Bexxar ? mouseCD20 follicular lymphoma Tovetumab mab human CD140a cancer Tralokinumabmab human IL-13 asthma etc. Trastuzumab Herceptin mab humanized HER2/neubreast cancer Trastuzumab Kadcyla mab humanized HER2/neu breast canceremtansine TRBS07 Ektomab 3funct ? GD2 melanoma Tregalizumab mabhumanized CD4 ? Tremelimumab mab human CTLA-4 cancer Trevogrumab mabhuman growth muscle atrophy due differentiation to orthopedic factor 8disuse and sarcopenia Tucotuzumab mab humanized EpCAM cancer celmoleukinTuvirumab ? human hepatitis B virus chronic hepatitis B Ublituximab mabchimeric MS4A1 cancer Ulocuplumab mab human C-X-C chemokine hematologicreceptor type 4 malignancies Urelumab mab human 4-1BB cancer etc.Urtoxazumab mab humanized Escherichia coli diarrhoea caused by E. coliUstekinumab Stelara mab human IL-12, IL-23 multiple sclerosis,psoriasis, psoriatic arthritis Vandortuzumab mab humanized STEAP1 cancervedotin Vantictumab mab human Frizzled receptor cancer Vanucizumab mabhumanized angiopoietin 2 cancer Vapaliximab mab chimeric AOC3 (VAP-1) ?Varlilumab mab human CD27 ? Vatelizumab^([) mab humanized ITGA2 ?Vedolizumab mab humanized integrin α₄β₇ Crohn's disease, ulcerativecolitis Veltuzumab mab humanized CD20 non-Hodgkin's lymphoma Vepalimomabmab mouse AOC3 (VAP-1) inflammation Vesencumab mab human NRP1 ?Visilizumab Nuvion mab humanized CD3 Crohn's disease, ulcerative colitisVolociximab mab chimeric integrin α₅β₁ solid tumors Vorsetuzumab mabhumanized CD70 cancer mafodotin Votumumab HumaSPECT mab human tumorantigen colorectal tumors CTAA16.88 Zalutumumab HuMax- mab human EGFRsquamous cell EGFr carcinoma of the head and neck Zanolimumab HuMax-CD4mab human CD4 rheumatoid arthritis, psoriasis, T-cell lymphoma Zatuximabmab chimeric HER1 cancer Ziralimumab mab human CD147 (basigin) ?Zolimomab mab mouse CD5 systemic lupus aritox erythematosus,graft-versus-host disease

In another embodiment using photoacoustic/thermoacoustic technology, thecirculating tumor, exosomes, or extracellular vesicles in the blood arequantified non-invasively by having a thermal energy source such aslaser microwave, RF, or other unit mounted on the patient's wrist, neck,etc. and a receiver to count and record the sound wave generated bycirculating cells to which the antibody-coated nanoparticles areattached.

In another embodiment, the ultrasonic receiver of the photoacoustic unitis an array of ultrasonic receivers mounted on a hand held probe. Thehand held probe contacts the patient's skin via a gel placed over thearea suspected to contain a tumor or lesion. It simultaneously recordsmultiple photoacoustic signals from the lesion during thermal energyapplication. Thermal energy applied pulses can range from one per secondto a million times or more per second. Each time a thermal pulse reachesthe nanoparticles, the nanoparticles expand and create aphotoacoustic/thermoacoustic response that is recorded by thephotoacoustic receiver.

The probe can be moved in any direction, e.g., up and down, side toside, etc., over the skin while recording the sound waves from thenanoparticles. Using a processor in the photoacoustic/thermoacousticunit, one uses the photoacoustic response data to construct a two- orthree-dimensional image of the tumor. The hand held probe permitsscanning any bodily surface, including but not limited to breast, eye,CNS, spinal cord, extremities, internal organs, eye, nose, chest,trachea, throat, abdomen, and urogenital organs. The data from theultrasonic array probe of the photoacoustic/thermoacoustic unit isstored in a computer during the probe's motion, permitting videoconstruction showing tumor shape, structure, location, etc. for videopresentation, evaluation, and archiving.

In one embodiment, the unit is capable of storing vast quantities ofdata from photoacoustic signals. The unit is also capable of storingvast quantities of data from non-stationary tissues, e.g., circulatingtumor cells and exosomes in blood vessels, that have accumulatedantibody coated nanoparticles on their cell membranes. The targetedcells can also be any normal or abnormal circulating cell in the bloodor lymphatic system. The photoacoustic unit reproduces signals fromthese mobile cells and/or exosomes as photoacousticcinematography/angiography or video.

In one embodiment, the cinematography or video recording is done by thephotoacoustic unit recording at least 30 frames/second of photoacousticsignals, and converting them into an image of a moving object. Acinematography or video is performed by obtaining at least 30 frames ofphotos of a moving object per second. In photoacoustic videography orphotoacoustic angiography, 30 or more frames of pulse signals from theheated nanoparticles per second are needed to reproduce or convert thestill images to a moving object, e.g., blood flow, etc. by the unit. Useof such a system is known: Peyman et al. Ophthalmic Surg Laser Imaging43 (2012) 143-51 doi: 10.3928.15428877-20120105-01 showing, however,lower resolution because no nanoparticles or photoacoustic imagingsystem was employed, and expressly incorporated by reference herein inits entirety.

In one embodiment the photoacoustic processor converts the microscopicstill images to a video or photoacoustic angiography; since the onlymoving parts in the vessels that are targeted with antibody coatednanoparticles are the circulating tumor cells or exosomes, extracellularvesicles or bubbles covered with antibody coated nanoparticles that areheated by a pulse of thermal energy produces an internal ultrasonicpulse signal recorded by the photoacoustic receiver. A moving image ofthe cells and exosomes can be created by the unit whether the cells areon the tumor interior or exterior.

Nanoparticle assisted photoacoustic video-angiography or nanoparticleassisted photoacoustic cinematography is novel and inventive. All“photoacoustic” terminology has previously been used for describingtissue heating or the difference in the temperature between two tissues,vessels vs. skin, and has been done with light alone, not in combinationwith nanoparticles with cell penetrating peptide (CPP), activating CPP(ACPP), biotin, streptavidin. In one embodiment, the method is performedfor therapy by providing the patient with at least one antibody-coatedfunctionalized nanoparticle having a detectable property, with theantibody targeting the functionalized nanoparticle to a specific patientsite, then heating the nanoparticles to generate a photoacoustic signal,i.e., thermal therapy, and imaging to visualize any localizednanoparticle at the site. The ultrasonic receiver of the photoacousticunit is an array of ultrasonic receivers mounted on a hand held probesimultaneously recording multiple photoacoustic signals from the lesionduring thermal energy application which in one embodiment is pulsating.The array of ultrasonic receivers of the photoacoustic unit mounted on ahand held probe simultaneously records multiple photoacoustic signalsfrom the lesion or vessels during thermal energy application,reproducing motion of moving nanoparticles and/or cells as ananoparticle assisted photoacoustic video-angiography or nanoparticleassisted photoacoustic cinematography.

In another embodiment, software associated with thephotoacoustic/thermoacoustic unit can enhance either or both thephotoacoustic signals and resulting images. Enhancement may facilitatedifferentiating exosomes from circulating cells due to the smallerexosome size. All exosomes or other types of extracellular vesicles areless than one micron; in contrast, tumor cells are five to twenty timeslarger than exosomes. The inventive system for the first time permits invivo observation and separation of exosomes from tumor cells, andseparation of circulating tumor cells from a tumor mass. The separatedcells or cell structures can be observed, counted, and quantified toassess the therapeutic effect of a procedure on tumor cells.

In another embodiment, after imaging and therapy, the biomarkers arecollected from liquid biopsies and compared with those obtained prior totherapy in different post-operative periods to confirm the therapeuticeffect of the procedure and prognosticate the condition.

In another embodiment, the antibody coated nanoparticles are conjugatedand administered with checkpoint inhibitors along with known immunetherapy agents and vaccines to facilitate circulating killer cellsattack and removal of tumor cells.

In another embodiment, the vaccines with or without VLP facilitatecirculating killer cells attacking and removal of tumor cells, and theantibody coated nanoparticles are administered with checkpointinhibitors, such as PD-1, PD-L1, CTLA-4, Jagged 1 inhibitor 15D11, etc.and Rock inhibitors (e.g., Fasudil or Botox) or Wnt inhibitor, such asniclosamide, ivermectin, etc., and for the future management of thetumor recurrences in the patient or treatment of metastatic disease.

In another embodiment, polymeric nanoparticles or polysaccharide orsynthetic polymers or porous silicon nanoparticles and/or microparticlesor magnetic luminescent porous silicon nanoparticles and/ormicroparticles or antibody coated nanoparticles conjugated with(alpha)-cyclodextrin, (beta)-cyclodextrin, (gamma)-cyclodextrin,hydroxypropyl-b-cyclodextrin (bHPCD) conjugated with biomarkers areadministered to enhance a vaccination effect and are taken up by antigenpresenting cells.

In one embodiment, genetic analysis of the patient is performed todetermine a sequence of the gene that is mutated. A sample of thepatient's blood is analyzed for any of the following indicia of thepresence or a neoplasm or a predisposition to a neoplasm: specific tumorbiomarker(s), non-specific tumor biomarker(s), extravascular vesicles,circulating tumor cells, tumor micro RNA, micro DNA, or any other tumorindicator. RNA sequencing reflects the dynamic nature of gene expressionfor detection of RNA fragments, including mRNA, noncoding RNA, chimericRNA, pathogen RNA, extracellular RNA, etc.

Examples of biomarkers have been previously disclosed. Other biomarkersinclude DNA hypermethylation, the presence of ZNF154 in colon, lung,breast, stomach, and endometrial tumor, and the stem cell marker NANOG,a mitochondrial oxidative phosphorylation/fatty acid oxidation moleculein highly malignant tumor-initiating stem-like cells (TICs) thatreprograms mitochondrial metabolism.

Use of results from a patient's genetic analysis advantageously permitsselection of a therapeutic agent, along with antibody-coatednanoparticles conjugated with porous silicon nanoparticles and/ormicroparticles or thermosensitive polymers and thermotherapy, to providethe greatest efficacy against cancers that are smaller than 4 mm indiameter. In general, such cancers have not grown to a size whereby theyshow genetic differentiation of the cancer cells. Treatment of thesesmall cancer cells can thus include treatment of the cancer stemcell(s). In one embodiment, nanoparticles with cell penetrating peptide(CPP), activating CPP (ACPP), biotin, streptavidin activated byelectromagnetic radiation, either in vitro or in vivo, enhance both genetransfer and cell proliferation of any desired cell, including stemcells, and by the using CRISPR-cas9 mediated Homology-IndependentTargeted Integration (HITI) or Homology Directed Repair (HDR).

In one embodiment, the patient's blood is processed to isolate thepatient's own natural killer (NK) cells, i.e., a type of lymphocyte thatis part of the patient's innate immune system, and dendritic cells,i.e., immune cells that process antigen material and present it on thecell surface to T cells of the immune system). NK cells and dendriticcells are isolated from a patient's blood using commercially availablekits known in the art, e.g., EasySep™ and RosetteSep™ STEMCELLTechnologies Inc., Tukwila Wash.; NK Cell Isolation Kit, MeltinyiBiotech, Bergisch Gladbach Germany. The natural killer cells/dendriticcells are rendered sensitized to the tumor in vitro. Sensitization isaccomplished by co-culturing the patient's natural killer cells and/ordendritic cells with IL-2 and the antibody-coated nanoparticlescontaining the optional penetration-enhancing agents and/orthermosensitive polymers as previously described. The patient'ssensitized natural killer cells/dendritic cells are then administered tothe patient at intervals to provide a booster immune therapy, much as avaccine booster injection does. The vaccine may be administered with orwithout VLPs. IL-2, IL17 is a protein produced by the T cells. When IL-2is conjugated with the thermosensitive antibody coated nanoparticle, andadministered with checkpoint inhibitors, such as PD-1, PD-L1, CTLA-4,Jagged 1 inhibitor, 15D11, etc. and Rock inhibitors, such as Fasudil orBotox, or Wnt inhibitors, such as niclosamide, ivermectin, andSelamectin and alpha lipoidic acid (ALA) or small molecule Wntinhibitors upon controllable temperature release, IL-2 is systemicallyavailable to enhance a T-cell response in the patient by cellsensitization and proliferation as a vaccine or treatment of metastaticdisease.

Thermal damage to the tumor cell membrane as a part of nanoparticleassisted thermotherapy releases antigens that, in vivo, activate andstimulate a dendritic cell immunogenic response. The activated dendriticcells induce a signal that additionally activates T cell-driven tumorcell damage or killing.

In one embodiment, the medium used to culture NK/dendritic cellscontains viral like particles (VLP), immune stimulators with cellpenetrating peptide (CPP), activating CPP (ACPP), biotin, streptavidinor antibody coated nanoparticles conjugated with (alpha)-cyclodextrin,(beta)-cyclodextrin, (gamma)-cyclodextrin, hydroxypropyl-b-cyclodextrin(bHPCD). The NK/dendritic cells pick up the VLP or immune stimulationwith toll like receptor 7/8 or interferons antibody coated nanoparticlesdendrimers and enhance sensitization against the tumor. If tumor cellbiopsy specimens are available, NK cells/dendritic cells are culturedfrom these biopsy specimens which additionally contain tumor lysate,killed circulating tumor (CT) cells, and their extracellular vesicles(ECV). In one embodiment, porous silicon nanoparticles and/ormicroparticles or other nanoparticles, with thermosensitive polymers andconjugated with tumor antibody and VLP, immune stimulation with tolllike receptor 7/8 or interferons antibody coated nanoparticles,dendrimers are administered to the patient intravenously, as the firststep of tumor vaccination and therapy. The nanoparticles become attachedto the tumor cells within a few minutes after administration.

In one embodiment, the tumor biomarkers from a patient's blood areidentified, and anti-tumor antibodies are prepared, using conventionalantibody techniques known in the art. The antibodies may be monoclonal,polyclonal, humanized, etc.; tumor antibodies also includes aptamers(oligonucleotide or peptides that bind to a specific target). Theantibodies/aptamers are then coated on diagnostic or therapeuticnanoparticles or quantum dots, and conjugated with checkpoint inhibitorssuch as PD-1, PD-L1, CTLA-4, Jagged 1 inhibitor 15D11, etc. and Rockinhibitors, such as Fasudil, hydroxyl Fasudil, etc., botox, 3C exoenzymeetc. or Wnt inhibitors, such as niclosamide, as a vaccine or treatmentof metastatic disease, which are then systemically administered to thepatient. In vivo, the tumor-antibody-coated nanoparticles seek the tumorcells via the specificity of the anti-tumor antibody component. In oneembodiment, adding a cell penetration enhancing agent to the polymer orother coating facilitates penetration of the tumor-antibody-coatednanoparticles into a tumor cell. Cell-penetration enhancing agentsrender the nanoparticle complex more biocompatible, and have beenpreviously disclosed; they include cell penetrating peptide (CPP),activated CPP (APCC), (poly)ethylene glycol (PEG), biotin streptavidin,etc.

In one embodiment, as previously disclosed, the tumor-antibody-coatednanoparticles are also coated with a thermosensitive polymer thatdissolves at a particular temperature, e.g., a polymer such as chitosanthat dissolves at a temperature of 40° C.-43° C., and/or an argininerich polymer, etc.

This coating, in addition to its thermosensitive properties with cellpenetrating peptides (CPP), activating CPP (ACPP), biotin, streptavidinor antibody coated nanoparticles conjugated with (alpha)-cyclodextrin,(beta)-cyclodextrin, (gamma)-cyclodextrin, hydroxypropyl-b-cyclodextrin(bHPCD), is administered with checkpoint inhibitors such as PD-1, PD-L1,CTLA-4, Jagged 1 inhibitor 15D11, etc. and Rock inhibitors, such asFasudil, Botox, 3C exoenzyme, etc., or Wnt inhibitor, such asniclosamide, etc., and includes one or more medicaments, genes, etc.thus providing additional therapy to the patient upon administration andthermotherapy as a vaccine or treatment of metastatic disease. In oneembodiment, adding a phospholipase, anti-phospholipid antibody, toxin(snake, scorpion, bee venom, etc. to the polymer or other coatingenhances the damage to the cell membrane from an anti-tumor antibodycoated nanoparticle. This beneficially increases the hyperthermal damageto cancer and other undesirable cells due to toxin release from thenanoparticles' coating of thermosensitive polymer at 40° C.-43° C.

In one embodiment, genes are provided that have a stimulatory action inresponse to light or ultrasound. An example of such a gene is the opsingene and members of the opsin family. In this embodiment, such genes areprovided to regulate cell membrane polarization and depolarization. Suchgenes can thus controllably create an action potential in the membraneof an excitable cell, such as a retinal cell, or a non-excitable cellsuch as a tumor cell. Controllable regulation may drive a permanentdepolarization state to render the cells accessible to a desiredmedicament for cell destruction.

In one embodiment, combinations of genes can be used for controllableregulation. As an example, genes responding to light to produce actionpotential, combined with genes that can modifying a defective gene(s) inthe cells of an organ, e.g., eye, brain, lung, spinal cord, peripheralnerve, lung, digestive tract, can be used in combination to facilitateregulation of actions including swallowing, breathing, gland secretion,etc., to restore the normal function of the organ. As another example,genes responding to light to produce an action potential, combined withinhibitory genes such as siRNA, RNAi, microRNA, can be used to inhibittumor function by simultaneous depolarization of the tumor cells. Thesegenes can additionally be combined with chemotherapeutic agent to worksynergistically and damage the tumor cells.

Systemic administration of tumor antibody coated nanoparticles, coatedwith thermosensitive polymers and a cell penetration facilitating agent,targets the nanoparticles toward the tumor cell membrane. Externalenergy is applied by a thermal delivery device that uses energy(electromagnetic radiation, microwave radiation, radiofrequency waves,an alternating magnet, focused ultrasound, etc.) to increase thetemperature of the nanoparticle. The heated nanoparticle absorbs moreenergy than the tissue surrounding the nanoparticle. The temperatureincrease causes the nanoparticles to expand. Expansion of thenanoparticles creates a photoacoustic, thermoacoustic, or ultrasoundwave, whose sound wave amplitude correlates with the amount of thetemperature increase, i.e., the degree of the temperature rise.

In one embodiment, the ultrasound wave is recorded by a transducer andis transmitted to a unit to image the nanoparticle increase intemperature as one-, two- or three-dimensional images. This unit isconnected to the thermal delivery device via a computer to maintain theamount of thermal energy needed for the time required to heat thenanoparticles to the desired temperature and for the desired time periodand thus release medicament(s), gene(s), CRISPR cas 9, to modify themutated genes by using CRISPR-cas9 mediated Homology-IndependentTargeted Integration (HITI) or Homology Directed Repair (HDR), VLP,immune stimulation, toll like receptor 7/8 or interferons antibodycoated nanoparticles, dendrimers, etc. to stimulate immune response.These agents may also be against microorganisms, e.g., bacterial, viral,fungal, or parasitic agents, which have developed resistance to thetherapeutic agents. For example, heated bacteria become more permeableto diffusion of appropriate medication; in contrast, non-heated bacteriaremain resistant.

In one embodiment, nanoparticles coated with the desired antibody (e.g.,anti-tumor antibody, anti-bacterial protein antibody, etc.) areadministered to the patient to assure that the antibody-nanoparticlecomplex is in contact with the appropriate cells or tissues. It will beappreciated that the appropriate cells or tissues may include bothcirculating cells (e.g., ECV, endosomes, leukemic cells, etc.) andnon-circulating cells (e.g., solid tumor).

In one embodiment, a small hand held photoacoustic unit with a smallthermal delivery unit e.g. laser, microwave, or radiofrequency unit isplaced externally over a subcutaneously located vessel to deliver apulse of energy and to heat the nanoparticles attached to thecirculating tumor cells and create a photoacoustic sound as they heatup. This records the sound wave each time a tumor cell passes by theexternal hand held unit, adjusts the temperature from 37° C.-43° C.,thus assessing and quantifying non-invasively the circulating tumorcells using the hand held thermal imaging device.

In one embodiment, photoacoustic technology imaging is controlled to alow temperature of 37° C.-43° C., thus assessing and non-invasivelymeasuring circulating cells using a hand held thermal imaging device.Imaging may be used in combination with any standard method, includingbut not limited to radiography, computed tomography (CT), magneticresonance imaging (MRI), ultrasound, positron and other molecularimaging devices.

In one embodiment, nanoparticles are conjugated with VLP derived fromplant viruses. In this embodiment, the VLP are used for cancer therapyby carrying sRNA, RNAi, etc. The host of these viruses are plants e.g.tobacco mosaic virus (TMV), bean yellow dwarf virus (BeYDV), etc., whichcannot infect the patient. Thermal application of the antibody coatednanoparticles provides the control over when and where these particlesare released to provide maximum benefit in immunotherapy. The VLP aregenerally immunogenic and do not require adjuncts to induce an immuneresponse. These modified viruses are devoid of genetic components andcannot replicate in the body. However, if a specific gene of a specificprotein e.g. an antibody, is conjugated with them and injected in theplant, the modified viruses produce large amounts of the antibody orprotein in the plant, which can subsequently be extracted and used inhuman infective or non-infective diseases or to produce a vaccine totreat e.g. Alzheimer's disease etc. The thermal application at 41-43degrees C. damages the VLP or other viruses and prevents theirproliferation without reducing their immunogenicity. Once the antibodyis produced, it can be used in diagnosing or guiding treatment to theaffected area in combination with nanoparticles with or without VLP anddrug delivery, and administering them with Rock inhibitors, such asFasudil, botox, C3 exoenzyme, etc., or Wnt inhibitors, such asniclosamide to be used as a vaccine or for treatment of metastaticdiseases as needed.

With respect to a gene(s) present in the polymer coating, e.g., aninhibitory gene such as siRNA, siDNA, RNAi, CRISPR to reduce theexpression of checkpoint proteins by the tumor cells etc., or anappropriate checkpoint inhibitor, may be used with antibody coatednanoparticles with cell penetrating peptide (CPP), activating CPP(ACPP), biotin, streptavidin or antibody coated nanoparticles conjugatedwith (alpha)-cyclodextrin, (beta)-cyclodextrin, (gamma)-cyclodextrin,hydroxypropyl-b-cyclodextrin (bHPCD). Checkpoint inhibitors enhancecellular immune responses to tumor specific proteins in the cancercells, as previously disclosed. In one embodiment, checkpointinhibitors, such as PD-1, PD-L1, CTLA-4, Jagged 1 inhibitor 15D11, etc.and Rock inhibitors, such as Fasudil, botox or Wnt inhibitors, such asniclosamide, ivermectin, and Selamectin and alpha lipoidic acid (ALA) asa vaccine or for treatment of metastatic diseases needed or are combinedwith nanoparticle-assisted targeted immunotherapy as an adaptive T-celltransfer mechanism. These, along with the a CRISPR/cas9 or CRISPRinterference (CRISPRi) complex, may perturb or modify the tumor genes byusing CRISPR-cas9 mediated Homology-Independent Targeted Integration(HITI) or Homology Directed Repair (HDR).

With respect to medicament(s), the medicament(s) would be releasedlocally present in the polymer coating or pluralities of the antibodycoated nanoparticles during thermotherapy. For a medicament that is abiologic, local release permits agents to be concentrated at the desiredsite without being released systemically resulting in systemic toxicitythe medicament may otherwise cause. As one example, anti VEGF agents,TNF inhibitors, antineoplastic medications such as taxol,antimetabolites, anti-inflammatory agents, steroids, checkpointinhibitors, such as PD-1, PD-L1, CTLA-4, Jagged 1 inhibitor 15D11, etc.and Rock inhibitors, such as Fasudil, C3 exoenzyme, Botox, etc., or Wntinhibitors, such as niclosamide, ivermectin, Selamectin, and alphalipoidic acid (ALA), antibiotics, antiviral agent, etc. can be used as avaccine or for treatment of metastatic diseases and become localizespecifically at a tumor or other site at significantly higherconcentrations to stop tumor neovascular growth or damage the tumoretc., without causing the known systemic complications such as heartattack, intestinal bleeding, kidney disease, liver disease, orsuppressing the normal humoral or cellular immune response of the body,etc. as seen in routine chemotherapy or immune therapy. As anotherexample, release of phospholipase enzymes can create a hole in themembrane of tumors or other cells to provide or facilitate entry of amedicament(s) and/or gene(s) entry into a cell.

In the inventive precision nanoparticle assisted-thermotherapy imaging(NATTI), the temperature of the tumor cell to which the nanoparticle isconjugated is controllably precisely increased. The temperatureincreases (a) releases a medicament(s) and/or gene(s) from athermosensitive coating on the nanoparticle, and (b) enhancespenetration of the medicament(s) and/or gene(s) through the open poresof the tumor cell membrane. NATTI technology includes acomputer-controlled thermal energy delivery unit to ensure attainment ofa desired increased temperature of the tumor for achieving thetherapeutic goal. Controlling thermal energy delivery to achieve atemperature from 38° C. to 42° C. for drug delivery or more in thetumor-nanoparticle complex to a tumor, or to another tissue affected bya disease as directed by antibody binding to a corresponding antigen. Itwill be appreciated that the increased temperature may be maintained atthe controlled desired level for any desired time interval, e.g., up to1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 minutes, or evenlonger, depending upon the need.

Typically, normal healthy cell membranes are comprised of thephospholipids phosphatidylserine (PS) and phosphatidylethanolamine (PE),which are located within the inner membrane and oriented toward the cellinterior. However, for cancer cells, the orientations of PS and PE areflipped, so each oriented toward the cell exterior. The melting point ofPS and PE is about 65° C.-70° C. degree. When the nanoparticles areheated to this temperature, the exposed PS and PE lipids of the cellmembrane melt, and create a dehiscence in the cell membrane throughwhich chemotherapeutic agents can freely flow into the tumor cell,killing the tumor cell.

For a medicament that is a chemotherapeutic agent, local release permitsa higher concentration of a chemotherapeutic agent to be contained inthe polymer coating, i.e., a supratherapeutic concentration, because itis confined to a localized site and would not result in systemictoxicity, yet still would achieve a higher therapeutic level at thetumor site. In one embodiment, a known existing antitumorchemotherapeutic agent is administered at a concentration that exceedsthat of a concentration that would be administered under typicaltherapy, yet that does not result in patient toxicity. Similarly, onecan use a toxic medicament(s) to locally perturb a cellular metabolicpathway or a specific cell cycle, e.g., local tumor cell perturbation.Such agents are generally not administered intravenously or orallybecause of their serious or fatal systemic toxicity and side effects.The concentration of the chemotherapeutic medicament(s) delivered by theinventive method is, in general, high locally, but less the 1/1000 ofthe concentration that would be required if the medicament(s) were to beeffective if delivered systemically.

Use of precision nanoparticle assisted thermotherapy and imaging (NATTI)may be used to fine-tune the approach to perturb survival mechanisms oftumors or other pathological cells. It will be thus appreciated that theinventive method may be used as a rapid en mass treatment of a cancer oran organ. For example, it may be used as a preliminary treatment inadvance of other therapies that in general have severe debilitatingsystemic complications such as immune suppression, etc. and which maytake longer to obtain approval for their administration. Thus,nanoparticle assisted thermotherapy and drug delivery NATTI avoids thechemotherapy complication of damaging the patient's immune system as aresult of one or multiple chemotherapeutic agents used in these latemetastatic cancer patients.

The nanoparticle assisted thermoacoustic imaging technology, with cellpenetrating peptides (CPP), activating CPP (ACPP), biotin, streptavidinalong with thermal energy, drives the medicament(s), gene(s), CRISPR cas9, VLP, immune stimulators combined with slow release polymers into thecancer cell, with simultaneous generation of an immune response to thecancer cell and inhibition of cancer cell proliferation by includingsiRNA, siDNA, etc. or RNAi, CRISPR to reduce the expression ofcheckpoint proteins by the tumor cells etc., along with simultaneouslyenhancing gene therapy by conjugating a CRISPR/cas9 or CRISPRi complexto the nanoparticle without relying on viral vectors by usingCRISPR-cas9 mediated Homology-Independent Targeted Integration (HITI) orHomology Directed Repair (HDR) to correct or inhibit a genetic componentof a tumor. Once inside a cell, e.g., a tumor cell or the cell of anorgan, the gene(s), along with the CRISPR/cas9 complex or CRISPRi, enterthe cell nucleus or mitochondria and precisely modify the gene pool ofthose cells. The RNA-guided bacterial endonuclease Cas9 is the effectorprotein of the type II CRISPR/Cas9 system that detects and subsequentlygenerates a double-strand break (DSB) in target DNA. This may treat adisease caused by a gene deficiency, or add a new useful gene(s), orremove and possible replace a gene, in the cell nucleus or themitochondria by the using CRISPR-cas9 mediated Homology-IndependentTargeted Integration (HITI) or Homology Directed Repair (HDR).

The gene(s) and/or medicaments(s) may be delivered to a specific site,but not released in the circulation from the nanoparticles untilreaching the required elevated temperature and after attaching to thetumor or other desired cells or desired location. It will be appreciatedthat the inventive method can be used in therapy for non-neoplasticdiseases. As one example, the amyloid plaques present in Alzheimer'sdisease may be used to produce anti-amyloid plaque antibodies andtreated by the inventive method. As another example, bacteria inpatients with severe sepsis refractory to standard antibiotic therapy,e.g., patients with methicillin-resistant Staphylococcus aureus (MRSA)etc., may be used to produce anti-bacterial antibodies and treated bythe inventive method. In this example, the method may be combined withextracorporeal treatment of blood, using a thermal energy delivery unitto provide electromagnetic radiation, radiofrequency waves, microwaves,focused ultrasound, an alternating magnetic field, etc., under thecontrol of the described NATTI unit, to controllably achieve atemperature of 42° C.-45° C. to kill the bacteria prior to cooling theblood to the normal 37° C. prior to reinfusion to the patient.

In addition, increasing the temperature of the nanoparticlesincrementally from 37° C.-43° C. allows precision nanoparticle assistedthermotherapy and imaging (NATTI) to release a gene(s) or medicament(s)from the nanoparticles. It will be appreciated that the methodbeneficially permits imaging a tumor or other desired cells, such asAlzheimer's plaques, that are present in a small lesion otherwiseinvisible by conventional imaging methods such as radiography.

The immune response is generated by two different mechanisms. Onemechanism is by releasing the VLP, immune stimulation with IL2, IL 17,toll like receptor 7/8 or interferons antibody coated nanoparticles ordendrimers with cell penetrating peptide (CPP), activating CPP (ACPP),biotin, streptavidin or antibody coated nanoparticles conjugated with(alpha)-cyclodextrin, (beta)-cyclodextrin, (gamma)-cyclodextrin,hydroxypropyl-b-cyclodextrin (bHPCD), which then stimulates a cellularimmune response at the site of the tumor. The other mechanism is by thethermotherapy-damaged tumor cells releasing their antigenic material inand beyond the surrounding tissues, creating a more active cellularimmune response due to the additional tumor antigens present, anddrawing the patient's immune cells, dendritic cell, T-lymphocytes,B-lymphocytes, macrophages, etc., to the tumor location. This mechanismalso advantageously provides immune memory functioning as an internalvaccination method. Specifically, local release of antigens from damagedtumor cells enhances a patient's immune response to a large amount ofother tumor cell associated antigens, creating a form of in vivovaccination. Such vaccination can be provided as needed, e.g., annually,at specific intervals, upon specific events, etc. to prime the patient'simmune cells against any future tumor cells, and protects againstreappearance of any tumors with similar antigenic components. Forexample, vaccination may be administered annually or biannually orbetween annual and biannual administration indefinitely, unless newbiomarkers are discovered in the patient, necessitating additionaland/or earlier therapy.

In addition, the inflammatory process created as a result of the immunetherapy and cellular response increases the temperature of the tissueinvolved, which is also recorded using photoacoustic technology imagingto image the tumor location and its potential metastatic lesionsanywhere in the body.

This embodiment results in precise, local, internally-inducedimmunotherapy and simultaneous vaccination. The antigen, e.g. VLP,immune stimulation with IL 3, toll like receptor 7/8 or interferonsantibody coated nanoparticles, dendrimers are delivered intravenouslywith thermosensitive polymers conjugated with antitumor antibody coatednanoparticles. The VLP and immune stimulators/thermosensitive polymersare released from these nanoparticles only when the temperature of thenanoparticle is increased to 40-43 C. degrees, and the nanoparticles arelocalized only at the tumor site, due to the specificity of theanti-tumor antibody with which the nanoparticles are conjugated.

As previously disclosed, various nanoparticle types, compositions,configurations, etc. are possible, including the following non-limitingexamples: organic, synthetic, metallic, non-metallic, magnetic,non-magnetic, paramagnetic, etc., configurations such as a nanosphere,nanotube, nanoshell, nanocage, nanocarbon, etc., including quantum dots,dendrimers, liposomes, or solid lipid nanoparticles, piezoelectricnanoparticles, etc.

In one embodiment, piezoelectric nanoparticles are stimulated by anultrasonic unit, providing a therapeutic effect by inducing an electriccurrent in cells. Depending upon the frequency, this exposure can killcells on one hand, or it can enhance growth of specific cells on theother hand. Application of thermal energy at a frequency in the range of1 Hz-20 Hz promotes cell growth. Application of thermal energy at afrequency greater than about 60 Hz damages cells. Cell death isdesirable for treating pathologies such as cancer. However, cellproliferation is desirable to facilitating tissue regeneration. Forexample, in this embodiment, a patient with a stroke, or a myocardialinfarction, or a spinal cord injury, may be treated to regenerate brain,heart, nerve tissue respectively. In this embodiment, the antibody usedis targeted to the damaged cells, i.e., neurons, cardiac cells, etc.,and treatment is with a pulsed frequency of 1 Hz-20 Hz or more isprovided for 1 min-10 min. It will be appreciated that this embodimentpermits stem cells to be controllably either stimulated or inhibited.

In one embodiment, the nanoparticle stimulates proliferation of in vitrocultured cells when the nanoparticle is exposed to and absorbs lightpulses of low frequency, i.e., frequencies in the range of 1 Hz-20 Hz.Conversely, in one embodiment, the nanoparticle inhibits cellproliferation when the nanoparticle is exposed to and absorbs lightpulses of very high frequency, i.e., frequencies in the range of >30Hz-100 Hz). Thus, selecting the frequency of the thermotherapy, and thusthe frequency to which the tumor antibody-coated nanoparticles or solidlipid nanoparticles are exposed, adds to the mechanisms of therapy thepatient receives if the light pulses are at low frequencies, i.e., nohigher than about 20 Hz.

In one embodiment, after sensitization of the immune cells with thetumor antigen, functionalized quantum dots with antibody coated againstcell membrane of immune cells is added so that the cell membranes of theimmune cells carry a marker that can be made visible with specificwavelength of light extracorporally.

In one embodiment, antibody coated nanoparticles or solid lipidnanoparticles are conjugated with thermosensitive polymers containingVLP/medication/genes, and administered with checkpoint inhibitors, suchas PD-1, PD-L1, CTLA-4, Jagged 1 inhibitor 15D11, etc. and Rockinhibitors, such as Fasudil or botox, or Wnt inhibitors, such asniclosamide etc., and intravenously administered to a patient at a farlower level than would be toxic to the body (i.e., 1/10 to 1/100 of thenon-toxic dose approved by the FDA). The VLP are released from thethermosensitive nanoparticles or solid lipid nanoparticles by thermalapplication at temperatures of 41° C.-43° C. The increase in temperatureis achieved initially or in the post treatment tissue using, e.g.,activation by light, electromagnetic radiation, microwave radiation,radiofrequency waves, focused ultrasound, or alternating magnetic fieldto preferentially heat the nanoparticles because of their high surfaceto volume ratio, and because the selected molecular composition of thenanoparticles or solid lipid nanoparticles preferentially absorbs morethermal energy than the surrounding normal cells. The tumor cells towhich the nanoparticles are attached are also heated. The thermal energymay also damage the VLP without reducing its immunogenicity at atemperature or 41-43 degrees C. so that there is no chance ofmultiplication of the VLP in the body.

In one embodiment, the increase in the temperature of the nanoparticlesor solid lipid nanoparticles results in their thermal expansion. Thermalexpansion of the nanoparticles produces an ultrasonic wave that passesthrough the body, is captured by a receiver, the ultrasonic pulse isconverted and amplified by an ultrasonic, photoacoustic, orthermoacoustic unit, imaged as a thermoacoustic signal or asnanoparticle assisted thermoacoustic signal, and converted by a computerto images, in one-, two-, or three-dimensions, of the temperature andthe lesion.

In one embodiment, the photoacoustic or nanoparticle assistedthermoacoustic unit controls the thermal energy delivery unit via aprocessor to maintain the temperature of the nanoparticles or solidlipid nanoparticles at a predetermined temperature as a closed circuitonce the nanoparticles have attached to the tumor cells. An increase inthe temperature to which the nanoparticles are exposed, i.e., at thenanoparticle level, from 37° C. to 41° C.-43° C. melts thethermosensitive polymers coating the nanoparticles or solid lipidnanoparticles, releasing under control the conjugated VLP,medication/gene, CRISPR cas 9 which are attached to the thermosensitiveantibody coated nanoparticles without relying on viral vectors, locallyat the desired site. This method is particularly effective in smalltumors, i.e., tumors less than 4 mm in diameter, because the tumor stemcells are still present at the original tumor site and can besimultaneously either genetically modified by the gene(s) usingCRISPR-cas9 mediated Homology-Independent Targeted Integration (HITI) orHomology Directed Repair (HDR) or damaged, or killed and eliminatedbefore metastasis has occurred.

In one embodiment, a plurality of the antibody-coated nanoparticles orsolid lipid nanoparticles are injected into a patient's circulation withthe cultured and tumor-sensitized NK cells/dendritic cells to target thetumor. It will be appreciated that such thermal damage to tumor cells,and a NK cellular response, generates and releases relatively largequantities of lytic enzymes and other cellular contents. In the case ofsmaller tumors, the released substances are of smaller quantities, butfor larger tumors it become necessary to remove them from the blood, byplasmaphoresis, plasma exchange, etc. These substances released into apatient's circulation, may be thought of as cellular debris or detritus,to prevent an immunogenic or cytokine storm in the body.

In one embodiment, the patient receiving the inventive therapy undergoesplasmapheresis to remove, e.g., such cytokines, enzymes, dead cells,etc. from the circulation. Plasmaphoresis is a known method to removecomponents from blood plasma. Because the patient's plasma is treatedextracorporeally, then reinfused, in contrast to reinfusing onlycellular components of the patient's blood, plasmaphoresis alsobeneficially detoxifies the patient's plasma without compromising bloodvolume and with minimal or no fluid loss. This technique avoids theserious complications and side effects of simply returning the cellularcomponents of the blood to the patient. Additionally, all precautionsare observed to avoid hypotension and loss of calcium ions in theprocess of citrate anticoagulation that this procedure requires. Thepatient can be treated initially with presently available anticoagulantssuch as heparin, coumadin, etc., which can be immediately neutralizedpost-procedure. Neutralization uses standard techniques known in theart, such as calcium, etc. Hemofiltration treatment is performed withactivated carbon, treatment on non-ionic exchange resins, etc. forremoving free toxin and also toxin bound with plasma proteins, etc. asin renal dialysis methods. The process may be instituted or repeated asneeded, e.g., if the tumor reappears. The addition of Rock inhibitors orWnt inhibitors along with other therapeutic agents can reduce the overtinflammatory response seen in immune therapy or an auto immune disease.

In one embodiment, to prevent a severe autoimmune response after tumorimmunotherapy, one uses extracorporeal plasmapheresis. A strong pulse oflight energy is applied to a tube containing blood cells to achieve atemperature up to 60° C. to kill immune cells containing quantum dots.The blood is then passed through a dielectrophoresis system tocharacterize and remove dead or live T-cells, sensitized killer cells,and tumor cells, prior to re-infusing the same blood or performing ablood transfusion in the patient while simultaneously administeringimmunosuppressive agents, including a biologic, to reduce the severeautoimmune response often seen after tumor immunotherapy.

The size of the nanoparticle may vary, and may vary depending on thesite of therapy and imaging as well as other factors. In one embodiment,the nanoparticle size ranges from 1 nm to 999 nm or more. In oneembodiment, the nanoparticle size ranges from 1 nm to 20 nm, which isideal for use in the eye and central nervous system to permit thenanoparticle access to the intercellular space, and also ideal for renalclearance without generating systemic side effects. Nanoparticles havinga size less than 10 nm in diameter, and not bound to a tumor, i.e.,nanoparticles that are free in the circulation, undergo rapid renalelimination from the body within a few hours of administration. Onlynanoparticles attached to the tumor cells remain in the body. Thisresults in a novel form of simultaneous local thermotherapy andvaccination.

The localized thermotherapy component of the method damages the tumorcells, thus disseminating tumor cell-associated antigens into thecirculation, generating a cellular immune response to the various tumorbiomarkers that were originally present. This dual thermotherapy andcellular response augments the effect of both immunotherapy andthermotherapy. The inventive method augments immunotherapy methods thatrelied on T-cells that had been sensitized to just a few tumor markers,or that relied only on checkpoint inhibitors to prevent the tumor cells'sequestration from T-lymphocytes. Previous methods of tumor vaccinationused intradermal or subcutaneous antigen administration, with theantigen taken up by antigen presenting cells, e.g., dendritic cells, togenerate specific killer cells only at a location remote from thespecific tumor site. The inventive method augments the previousimmunotherapy methods by combining immunotherapy to act synergisticallywith thermotherapy locally at the tumor site, thus preventing exposureof the entire body to therapeutic agents or checkpoint inhibitors thatcause immune suppression or auto-immune disease.

In one embodiment, cultured killer cells sensitized to a tumor areadministered simultaneously with the anti-tumor antibody coatednanoparticle-conjugated VLP to attack the tumor cells by the patientst-lymphocytes etc. and remove the dead tumor cells. For example, anintradermally administered antitumor antibody-coated nanoparticle withVLP can be administered with checkpoint inhibitors such as PD-1, PD-L1,CTLA-4, Jagged 1 inhibitor 15D11, etc., and Rock inhibitors, such asFasudil, botox or Wnt inhibitors, such as niclosamide, in subsequentrounds of therapy during a postoperative period to induce an immuneresponse as needed. This embodiment decreases the likelihood of, orprevents potential recurrences of the tumor or treats a recurrence of atumor as a vaccination or therapy in recurrences. Since tumorrecurrences are generally non-sensitive to ordinary therapy, it might beimportant in some cases that vaccination is done along with thecheckpoint inhibitors to be able to attack the tumor cell recurrencesthat potentially have survived the previous therapy, in addition toproviding Rock inhibitors or Wnt inhibitors that reduce excessiveinflammatory disease and discourage tumor cell proliferation.

In one embodiment for use in larger tumors of a sufficient size forbiopsy, an antibody directed to the tumor lysate (TL) is used as asource of tumor-associated antigens (TAAs), and is conjugated with thenanoparticles or solid lipid nanoparticles for generating therapeuticanti-tumor immune responses. One can generate in vivo immunity againstmultiple TAA simultaneously from the killed or damaged tumor cellsduring the thermotherapy. This embodiment broadens the repertoire ofTAA-specific T-cell clones available for activation or therapy of thesetumors.

In one embodiment, after an initial thermotherapy procedure, a bloodsample is obtained from the patient. This blood sample contains releasedtumor antigens that are recoverable prior to treatment by the inventivemethod using various immunoassays or methods of searching forbiomarkers. The tumor antigens are then used to generate, in vitro,additional T-cells that are sensitized to many TAA for future use in,along with VLP for vaccination, and are administered with checkpointinhibitors, such as PD-1, PD-L1, CTLA-4, Jagged 1 inhibitor 15D11, etc.,and Rock inhibitors, such as Fasudil, etc. or Wnt inhibitors, such asniclosamide, etc. to the same patient to enhance the immune response asa vaccine or to treat potential recurrences of the same tumor.

In one embodiment, immunostimulatory oligonucleotide-loaded cationicgraphene oxide, carbon nanotube, gold/iron, iron/zinc oxide, or cadmiumsulfate nanoparticles are combined with photothermally enhancedimmunogenicity to achieve combined thermo-immune therapy. In oneembodiment, RNA oligonucleotides/graphene or graphene oxide, or longdouble stranded RNA/graphene oxide induces a controlledimmunostimulation in combination with oncogene silencing RNAi.

Nanoparticles, dendrimers, carbon nanotubes, lipid-based carriers,micelles, gold nanoshells/nanocages, PLGA, chitosan, PEI cationic lipid,and cationic polymers with cell penetrating peptides (CPP), activatingCPP (ACPP), biotin, streptavidin or antibody coated nanoparticlesconjugated with (alpha)-cyclodextrin, (beta)-cyclodextrin,(gamma)-cyclodextrin, hydroxypropyl-b-cyclodextrin (bHPCD) are usefulfor gene therapy, gene delivery, by using CRISPR-cas9 mediatedHomology-Independent Targeted Integration (HITI) or Homology DirectedRepair (HDR) and immunotherapy. These have the advantages of beingeasily prepared, biodegradable, non-immunogenic, non-toxic, and watersoluble.

EXAMPLE 1

T cells and dendritic cells are obtained from a patient's blood, andgrown in culture along with a tumor or other antigen, plus nanoparticlesor solid lipid nanoparticles coated with thermosensitive polymersconjugated with antigen and VLP using culture methods known in the art.

The nanoparticle complex is injecting them along with checkpointinhibitors and IL-2. The inventive method is applied, killing tumorcells, and increasing the response of T-cells and dendritic cell.

The patient's blood is assessed for new biomarkers from the dead cells.

The cultured T-cells and dendritic cells are harvested, along with thenanoparticle-coated antigen plus VLP or RNA or DNA phages. These arestored under appropriate conditions, and reinjected into the patientwith low dose coated nanoparticles or systemic medicaments to beadministered with checkpoint inhibitors such as PD-1, PD-L1, CTLA-4,Jagged 1 inhibitor 15D11, etc. and Rock inhibitors, such as Fasudil orWnt inhibitors such as niclosamide as needed, e.g., semi-annually,annually, biannually, etc. as a vaccination or in tumor recurrences inmetastatic disease, with repetition as needed. This is followed up withcounting or quantifying circulating DNA, exosomes or circulating cellsto recognize potential tumor recurrences.

EXAMPLE 2

A checkpoint inhibitors and rock inhibitors, e.g., Botox up to 50-100picograms (pg), or 100 picograms (pg) to 1 nanogram (ng) or more orfasudil from 100 picograms (pg) to 10 nanograms (ng) or 50 nanograms(ng) to 1 milligrams (mg) or Wnt inhibitors, e.g., niclosamide is addedto a thermosensitive polymer conjugated with antibody coated pluralitiesof nanoparticle for controlled release with the checkpoint inhibitorusing the inventive controlled thermotherapy, NATTI, to release themedication locally at temperature of 41-43 C. degrees along immunestimulators, such as VLP to treat a patient with breast, colorectal,glioblastoma, prostate, eye or skin melanomas, pancreatic, lung cancer,and/or ovarian cancer, etc. A checkpoint inhibitor, such as nivolumab orPD-L1, CTLA-4, Jagged 1 inhibitor 15D11, etc. and Rock inhibitors, suchas Fasudil or botox, or Wnt inhibitors, such as niclosamide, ivermectin,and Selamectin, and alpha lipoidic acid (ALA) is combined withpluralities of antibody coated nanoparticle assisted targetedimmunotherapy for adaptive T-cell transfer to overcome the limitationsof standard immunotherapy and prevent a cytokine storm. In oneembodiment the Rock inhibitor Fasudil can be taken orally at the dose of40-80 mg as needed and niclosamide 1-2 gram once or repeated in a week,ivermectin and/or Selamectin also can be given orally at a dose of 1gram orally for a period of time during and shortly after thermotherapyfor a few days as needed.

EXAMPLE 3

Nanoparticles or solid lipid nanoparticles are conjugated with achimeric receptor, a CD19 protein that is found only on B cells, alongwith the T-cells cultured in vitro that expresses a chimeric antigenreceptor (chimeric antigen receptor T (CAR T)-cells) to target abnormalB cells seen in leukemia along with PD-L1, CTLA-4, Jagged 1 inhibitor15D11, etc. and Rock inhibitors, such as Fasudil or botox, or Wntinhibitors, such as niclosamide. The reappearance of new biomarkers asneoantigens in these patients can be also treated in the postoperativeperiod using the inventive method repeated therapy as vaccination alongwith Rock inhibitor encourage the abnormal mature cells to undergoapoptotic degeneration rather cell proliferation with abnormal geneticchanges which is characterized by the tumors occurring as a result ofaging process and not a pre-existing genetic mutation by usingCRISPR-cas9 mediated Homology-Independent Targeted Integration (HITI) orHomology Directed Repair (HDR).

Plasmaphoresis is simultaneously performed or performed after treatment.

This example treats acute and chronic hematologic malignancies such asacute lymphoblastic leukemia, non-Hodgkin lymphoma, chronic lymphocyticleukemia, chronic myelogenous leukemia, etc.

EXAMPLE 4

Pluralities of antibody coated nanoparticles or solid lipidnanoparticles are conjugated with all-trans retinoic acid (ATRA) andarsenic trioxide to target leukemia cells in acute promyelocyticleukemia and used in the inventive method. The all-trans retinoic acidis released at the site of the tumor without exposing the entire body tothe toxic medication, simultaneously, plasmophoresis is performed toclear all toxin released in the blood, along with leukemic cells. It isappreciated that other blood cell cancers are removed in the samesession.

EXAMPLE 5

In a patient with a hematologic malignancy that is resistant tochemotherapeutic agents or immune therapy, NATTI is performed with genedelivery by using CRISPR-cas9 mediated Homology-Independent TargetedIntegration (HITI) or Homology Directed Repair (HDR), along withchemotherapeutic agents, to target all immune cells initially withoutsubjecting the patient to systemic heavy chemotherapy, followed by bonemarrow transplantation, without exposing the entire body to systemicchemotherapy.

EXAMPLE 6

Pluralities of antibody coated nanoparticles or solid lipidnanoparticles or antibody coated nanoparticles conjugated with(alpha)-cyclodextrin, (beta)-cyclodextrin, (gamma)-cyclodextrin,hydroxypropyl-b-cyclodextrin (bHPCD) are conjugated with RNA thatcontains an aptamer, ribosomes, and siRNA or RNAi, CRISPR to reduce theexpression of checkpoint proteins by the tumor cells etc., in athermosensitive polymer and administered to using NATTI to targetspecific tumor cells.

EXAMPLE 7

The microenvironment of the cancer cell is modified by deliveringmedicaments that block the uptake of exosomal signals and prevent theuptake of ECV. Such medications include choloroquine, heparin,cytochalasin D, and ethyl-isopropyl amiloride are conjugated withpolymeric coating and conjugated with antibody coated nanoparticles orsolid lipid nanoparticles administered to the patient and released withNATTI. These medications are approved for patient use. The medicamentsare provided using NATTI in conjunction with chemotherapeutic agents androck inhibitors.

EXAMPLE 8

The inventive method provides nanoparticle assisted localizedimmunothermotherapy and thermotherapy for delivery of customizedvaccines with or without VLP to target core mutations in a patient. Theimmune cells or T-cells that can attack those core mutations areidentified via a cancer biomarker. The immune cells or T-cells are thencultured with the nanoparticles or solid lipid nanoparticles coated withthermosensitive particles and VLP adjuvants and IL-2. The antibodycoated nanoparticles or antibody coated nanoparticles conjugated with(alpha)-cyclodextrin, (beta)-cyclodextrin, (gamma)-cyclodextrin,hydroxypropyl-b-cyclodextrin (bHPCD) with checkpoint inhibitors, such asPD-1, PD-L1, CTLA-4, Jagged 1 inhibitor 15D11, etc., and Rockinhibitors, such as Fasudil or Botox etc., or Wnt inhibitors, such asniclosamide, etc., are injected into the patient, controllably heatedusing a thermal energy source, and imaged, for specific patient, orthose with metastatic disease or recurrences as immunotherapy, such asin breast cancer, prostate cancer, glioblastoma, lung cancer, melanoma,ovarian cancer, pancreatic cancer, intestinal or colon cancer, etc.

EXAMPLE 9

Antibody coated nanoparticles or antibody coated nanoparticlesconjugated with (alpha)-cyclodextrin, (beta)-cyclodextrin,(gamma)-cyclodextrin, hydroxypropyl-b-cyclodextrin (bHPCD) areconjugated with RNA phage VLP, adjuvants, which is generally stable upto about 50° C. VLPs or adjuvants of the related RNA phage PP7 arecrosslinked with inter-subunit disulfide bonds, rendering themsignificantly more stable. They exhibit high immunogenicity. Suchnanoparticles complement the inventive NATTI technology and can beemployed in anti-cancer and antibacterial treatment. Lytic phages attachto receptors on the bacterial surface, inject their genetic materialthrough the bacterial membrane, and overtake the bacterium'stranscription and translation machinery to synthesize new phages. Theapplication of thermotherapy damages all VLP, phages, virocides orforeign protein and eliminate their future growth and potential adversereactions.

EXAMPLE 10

To prevent a severe autoimmune response after tumor immunotherapy,before extracorporeal plasmapheresis, one uses the nanoparticle assistedthermotherapy and imaging system to apply heavy thermal energy to a tubecontaining blood cells and to achieve a temperature as high as 60° C. tokill the sensitized immune cells containing nanoparticles or solid lipidnanoparticles. Blood is then passed through a dielectrophoresis systemto characterize and remove dead or live T-cells, sensitized killercells, and dead tumor cells prior to re-infusing blood in the patientwhile simultaneously administering immunosuppressive agents and Rockinhibitors, including biologics. This reduces the severe autoimmuneresponse often seen after tumor immunotherapy.

In one embodiment, pain is one of the most common symptoms ofinflammation in the patient; pain can be observed in viral or bacterialinfections, surgery or trauma, an auto immune response, e.g., rheumatoidarthritis, affecting the joints, spondylitis of the vertebrae, afterspinal cord injury, diabetic neuropathy, Fibromyalgia, after radiationor traumatic injuries, or radiation therapy and chemotherapy of themalignancies, the so called radiation or chemotherapy inducedneuropathy. Fibromyalgia is a chronic undefined disorder with thesymptoms of widespread pain, stiffness, fatigue, tenderness, anxietyand/or depression. It can be associated with, hypothyroidism,polymyalgia rheumatic, autoimmune rheumatoid arthritis, lupuserythematosus and other inflammatory disorders. There is no cure forfibromyalgia and treatment has been so far limited to analgesicsantianxiety/hypnotic agents, skeletal muscle relaxants antidepressants.In nerve injury, the aberrant nerve regeneration produces abnormalexcitability or sensitivity to various mechanical or thermal or chemicalstimulation and produces central sensitization and chronic painsensation. The peripheral nerve injury regardless of its origin such asradiation neuropathy or diabetic neuropathy affects the vascular supplyof the nerves and their glial cells which produce proinflammatorycytokines worsening the condition. In addition, the radiation inducesvascular occlusion in the tissue with subsequent ischemia, release ofTGF beta, oxygen free radicals (reactive oxygen species), inducingfibrous tissue proliferation and ultimately tissue fibrosis increasenerve compression and pain sensation. These phenomenon are a commoncomplication of external x-ray radiation, proton beam radiation or localradioactive cobalt plaque implantation etc. radiation. The higher theradiation dose, the more likelihood of developing radiation neuropathy.The changes, depending on the radiated area, are calledradiation-induced brachial plexopathy (RIBP), lumbosacralradiculo-plexopathy, acute lumbosacral plexopathy, post hysterectomy andradiation side effects, brachial neuropathy, peripheralneuropathy/fibrosis or radiation-induced peripheral neuropathy (RIPN),lid and conjunctival fibrosis and corneal scarring and cataractformation, lumbosacral radiculoplexopathy, and nerve trunk damage.Additional risk factors are diabetes mellitus or co-existence ofdiabetic neuropathy, arteritis, and collagen vascular diseases andsimultaneous chemotherapy administered for cancer therapy. Delayedeffects of radiation are extensive fibrosis within and surrounding nervetrunks, and ischemia by injury to capillary networks supplying thenerves, compensated for by neovascularization and nerve demyelination.Depending on the area that has been radiated one can observe, cranialnerve damage predominantly involving the optic nerve associated withacute loss of visual acuity, trigeminal neuropathy develops aftercavernous sinus tumor therapy, radiation injuries involving theglossopharyngeal nerve with swallowing impairment; vagus nerve afterthoracic radiation therapy for breast, upper limb injury with classicprogressive brachial plexopathy, axial neurological injury, delayedbrachial plexopathy with comorbidity of lymphedema such as in radicalmastectomy with extended lymph node dissection, and simultaneouschemotherapy with cisplatin or taxol the symptoms varies from decreasessensory perception, to loss of the function affecting both the sensoryand motor nerves with various degree of paresthesia, debilitating painand often limited motor function of the affected area because of tissuefibrosis and loss of the nerve function. The therapy of these seriouscomplications are in general limited, and a prophylactic treatment hasnot been done. The therapy has been limited to symptomatic treatmentincluding non-opioid analgesics, tricyclic antidepressants andanti-epileptics or benzodiazepines, restriction of aggravating factors,Carbamazepine, inhibiting voltage-gated sodium channels, reducing theexcitability of neural membranes, serotonin-norepinephrine reuptakeinhibitors, Clodronate, pregabalin, topical lidocaine, opioids,analgesics, Capsaicin, N-methyl-aspartate (DMDA) inhibitor. Othertopical agents such as amitriptyline, nifepidine, pentoxifyline,neuromodulators include electrical or chemical implantable andnon-implantable devices that can modulate the pain sensation or nerveelectrical signals. These require surgical implantation of the devicethat generates an electrical pulse that interferes with disease inducedabnormal pulses that transmit pain sensation as with deep brainstimulation. These devices require complex surgical procedures and withtime, the contact electrodes need to be changed, while the diseaseprocess continues to worsen despite these interventions.

The canonical Wnt/β-catenin plays an important role in the expression ofseveral inflammatory molecules during acute or chronic inflammatorydiseases affecting skin, mucosal surfaces, subcutaneous tissue, muscle,interstitial tissue of the body including the brain, lung, liver,intestine, genitourinary system, the conjunctiva, nasal, oral, andthroat including dry eye syndrome.

In one embodiment, for a patient suffering from pain after radiation orcombined with chemotherapy, the antibody coated nanoparticles with Wntinhibitors and/or Rock inhibitors are administered either locally, as anointment, suspension or injected to the area of radiation and neuronalpain; compounds are used, such as: FH535, IWP-2, PNU-74654, IWR-1endo.IWR-exo, Demethoxy Curcumin, CCTO36477, KY02111, WAY-316606, SFRP, IWP,LGK974, C59, Ant1.4Br/Ant 1.4Cl, Ivermectin, Niclosamide, apicularen,and bafilomycin, XAV939, XAV939, G007-LK and G244-LM, NSC668036,SB-216763, gemtuzumab, etc., small molecule Wnt inhibitor PKF118-310,the Wnt/β-catenin pathway inhibitor and fasudil, a rock inhibitorFasudil (HA-1077), a selective RhoA/Rho kinase (ROCK) inhibitor, orY-27632, a small molecule inhibitor of ROCK1 and ROCK2,Fasudil1-(5-Isoquinolinesulfonyl)-2 Methylpiperazine Calcium ChannelBlockers, Membrane Transport Modulators, etc., Canakinumab ivermectin,or niclosamide, Botulinum Botox, all having a good penetration into theskin or mucosa, subcutaneous tissue, muscle, interstitial tissue of thebody including the brain lung, liver, intestine, genitourinary system,intravenously or intra-arterially, or can be delivered as s slow releasecompound implanted or injected inside the tissue with any polymericcompound, such as the polymers previously disclosed (e.g.,polycaprolactone, poly(glycolic) acid, poly(lactic) acid, polyanhydride)or lipids that may be formulated as antibody coated microspheres orantibody coated dendrimers or antibody coated nanoparticles, includingquantum dots, nanotubes, or nanowires. As an illustrative example,Fasudil may be mixed with polyvinyl alcohol (PVA), the mixture thendried and coated with ethylene vinyl acetate, then cooled again withPVA. Niclosamide bound with liposomes may be applied topically, eitherin the form of drops or as an aqueous based cream, or may be injectedinto body cavities, intravenously, intra-arterially as antibody-coatednanoparticles and/or microparticles with (alpha)-cyclodextrin, or(beta)-cyclodextrin, or (gamma)-cyclodextrin,hydroxypropyl-b-cyclodextrin (bHPCD). In a formulation for topicalapplication, the drug is slowly released over time as the liposomecapsule degrades due to wear and tear from the surface of the skin ormucosa or in the tumor or interstitial tissue. In a formulation forinjection and nano solid lipids, or liposome capsule degrades due tocellular digestion, other slow release polymers such as PLA, PGA,Polycaprolactone, microsphere, dendrimers) or as nanoparticles and/ormicroparticles of porous silicon or with (alpha)-cyclodextrin, or(beta)-cyclodextrin, or (gamma)-cyclodextrin,hydroxypropyl-b-cyclodextrin (bHPCD) are also utilized, or some such asFasudil orally at doses of 40-80 mg. This is equal to 1 microgram/ml to40 microgram/ml or to 80 microgram/ml or more for topical applicationhaving ranges of 40 ng/ml to 4 micrograms/ml, or 0. 1 microgram/ml to 40microgram/ml or more for topical applications without having the sideeffects of steroid preparation, in addition the following compounds arereadily available and some have been approved by the FDA: potent ROCKinhibitor; orally bioavailable Fasudil hydrochloride, Inhibitor ofcyclic nucleotide dependent- and Rho-kinases GSK 269962, Potent andselective ROCK inhibitor GSK 429286, Selective Rho-kinase (ROCK)inhibitor H1152 dihydrochloride, Selective Rho-kinase (ROCK) inhibitorGlycyl H 1152 dihydrochloride, Selective Rho-kinase (ROCK) inhibitor;more selective analogue of H1152, Cell-permeable, selective Rho-kinaseinhibitor OXA 06 dihydrochloride, potent ROCK inhibitor PKI1447dihydrochloride, potent and selective ROCK inhibitor; antitumor SB772077B, potent Rho-kinase inhibitor; vasodilator SR 3677dihydrochloride, potent, selective Rho-kinase (ROCK) inhibitorTC-57001,potent and highly selective ROCK inhibitor; orally active Y-27632dihydrochloride, Botox or botulinum toxin as injectable preparation oftopical ointment or with slow release polymers described above.Available Wnt inhibitors include small molecule Wnt inhibitorPKF118-310, the Wnt/β-catenin pathway inhibitor, niclosamide, ivermectinetc.

In one embodiment, the antibody coated nanoparticles containing a coatedWnt inhibitor or rock inhibitor are administered locally as an ointment,or injected subcutaneously or intra-arterially, intravenously,interstitially to multiple areas, intramuscularly, by injecting asolution of 0.01-0.5 ml or more in a physiologic PH adjusted to 7-7.5 PHand osmolarity of 280-300 mOsm close to the major neurons on a patientsuffering from the symptom of inflammation. In the patient, pain can beobserved, in viral or bacterial infections, surgery or trauma, an autoimmune response, e.g., rheumatoid arthritis, affecting the joints,spondylitis of the vertebrae, after spinal cord injury, diabeticneuropathy, etc.

In one embodiment, the antibody coated nanoparticles containing a coatedWnt inhibitor or rock inhibitor are administered locally as ointment, orinjected subcutaneously or intra-arterially, intravenously,interstitially to multiple areas, intramuscularly, by injecting asolution of 0.01-0.5 ml or more in a physiologic PH adjusted to 7-7.5 PHand osmolarity of 280-300 mOsm close to the major neurons in a patientsuffering from the symptom of inflammation. In the patient, theinflammation and pain is occurring after radiation or traumaticinjuries, or radiation therapy and chemotherapy of the malignancies, theso-called radiation or chemotherapy induced neuropathy.

In one embodiment, the antibody coated nanoparticles containing a coatedWnt inhibitor or rock inhibitor are administered locally as ointment, orinjected subcutaneously or intra-arterially, interstitially to multipleareas, intramuscularly, using a fine needle, injecting a solution of0.01-0.5 ml or more in a physiologic PH adjusted to 7-7.5 PH andosmolarity of 280-300 mOsm close to the major neurons as prophylacticfor prevention of the radiation complications such as neuropathy andfibrosis in the areas to be irradiated or that will be in the path ofthe beam of radiation.

In one embodiment, the antibody coated nanoparticles containing a coatedWnt inhibitor or rock inhibitor are administered postoperatively locallyas an ointment, or injected subcutaneously or intra-arterially,interstitially, using a fine 23-32 gauge needle, injecting a solution of0.01-0.5 ml or more in a physiologic PH adjusted to 7-7.5 PH andosmolarity of 280-300 mOsm to multiple areas, intramuscularly, close tothe major neurons treated, the higher the radiation dose increased thelikelihood of developing radiation neuropathy at the radiated area tothe brachial plexus.

In one embodiment, the antibody coated nanoparticles containing a coatedWnt inhibitor or rock inhibitor are administered postoperatively locallyas an ointment, or injected subcutaneously or intra-arterially,interstitially to multiple areas, intramuscularly, using a fine 23-32gauge needle injecting a solution of 0.01-0.5 ml or more in aphysiologic PH adjusted to 7-7.5 PH and osmolarity of 280-300 mOsm closeto the major neurons treated higher the radiation dose after developingradiation neuropathy at the radiated area, close or to brachial plexusarea.

In one embodiment, the antibody coated nanoparticles containing a coatedWnt inhibitor or rock inhibitor are administered locally as an ointment,or injected subcutaneously or intra-arterially, interstitially tomultiple areas, intramuscularly, using a fine 23-32 gauge needleinjecting a solution of 0.01-0.5 ml or more in a physiologic PH adjustedto 7-7.5 PH and osmolarity of 280-300 mOsm close to the major neurons asprophylactic for prevention of the radiation complications, such asneuropathy and fibrosis in the areas to be irradiated or will be in pathof the beam of radiation.

In one embodiment, the antibody coated nanoparticles containing a coatedWnt inhibitor or rock inhibitor are administered locally as an ointment,or injected subcutaneously or intra-arterially, interstitially tomultiple areas, intramuscularly, using a fine 23-32 gauge needleinjecting a solution of 0.01-0.5 ml or more in a physiologic PH adjustedto 7-7.5 PH and osmolarity of 280-300 mOsm close to the major neuronsand the area of radiation induced vascular occlusion in the tissue withsubsequent ischemia, release of TGF beta, oxygen free radicals (reactiveoxygen species), inducing fibrous tissue proliferation and ultimatelytissue fibrosis, increased nerve compression and pain sensation wherethe Rock inhibitors, such as botox, inhibits TGF beta and subsequentfibrosis.

In one embodiment, the antibody coated nanoparticles containing a coatedWnt inhibitor or Rock inhibitor are administered locally as an ointment,or injected subcutaneously or intra-arterially, interstitially tomultiple areas, intramuscularly, using a fine 23-32 gauge needleinjecting a solution of 0.01-0.5 ml or more in a physiologic PH adjustedto 7-7.5 PH and osmolarity of 280-300 mOsm close to the major neuronsand the area of radiation at the lumbosacral radiculo-plexopathy, acutelumbosacral plexopathy, post hysterectomy and radiation sideinflammation and fibrosis, brachial neuropathy, peripheralneuropathy/fibrosis or Radiation-induced peripheral neuropathy (RIPN).

In one embodiment, antibody coated nanoparticles containing a coated Wntinhibitor or rock inhibitor are administered, using a fine 23-32 gaugeneedle injecting a solution of 0.01-0.5 ml or more in a physiologic PHadjusted to 7-7.5 PH and osmolarity of 280-300 mOsm to a patient who hasdeveloped radiation complications, such as neuropathy and fibrosis inthe areas that have been irradiated, or have been in the path of thebeam of radiation.

In one embodiment, antibody coated nanoparticles containing a coated Wntinhibitor or rock inhibitor are administered to a patient who hasdeveloped complication of the radiation neuropathy or fibrosis in theareas that have been irradiated or have been in the path of theradiation producing symptoms of pain and neuropathy, using a fine 23-32gauge needle and injecting a solution of 0.01-0.5 ml or more in aphysiologic PH adjusted to 7-7.5 PH and osmolarity of 280-300 mOsm.

in one embodiment, Rock inhibitors are administered with antibody coatednanoparticles conjugated with thermosensitive nanoparticles andAdalimumab, a humanized antibody, administered topically orsubcutaneously at a non-toxic dose.

In one embodiment, the Wnt inhibitors or Rock inhibitors areadministered with nanoparticles or dendrimers coated withthermosensitive polymers and conjugated with lactic or glycolic acid orcombinations thereof, or nanoparticles or microparticles of poroussilicon, and administered as drops or by injecting a solution of0.01-0.5 ml or more in a physiologic PH adjusted to 7-7.5 PH andosmolarity of 280-300 mOsm in a patient who has developed complicationof the radiation neuropathy or fibrosis in the areas that have beenirradiated or have been in the path of the radiation, producing symptomsof pain and neuropathy.

In one embodiment, Rock inhibitors are administered or injected using asolution of 0.01-0.5 ml or more in a physiologic PH adjusted to 7-7.5 PHand osmolarity of 280-300 mOsm with antibody coated nanoparticles,dendrimers, liposomes, solid lipid nanoparticles, micro- ornanoparticles of porous silicone, etc. to a patient who has developedcomplications of radiation neuropathy or fibrosis in the areas that havebeen irradiated or have been in the path of the radiation, producingsymptoms of pain and neuropathy.

In one embodiment, a formulation of Wnt or Rock inhibitors used to treatskin, mucosal lichen planus, as well as other conditions, is disclosed.Rock inhibitors and Wnt inhibitors are used as topical drops, sprayapplications, or injections into the lesion or surrounding tissue, or anas implantation in or on the in the tissue. For example, a topicaladministration may contain between about 10 pg/ml drug to about 50.mu.g/ml drug in a formulation which may be applied at bed time orthroughout the day. For injection, a dose of about 50 pg/ml to about 200.mu.g/ml may be used as a surgical implant, for example, in a diffusiblewalled reservoir close to the major nerve in the treated area, or may becontained within an inert carrier such as microspheres or liposomes,solid lipid nanoparticles of nanoparticles of porous silicon to providea slow-release drug delivery system to the radiated areas.

In one embodiment, a formulation of nanoparticles coated with Wnt orRock inhibitors is used from the group consisting of topicaladministration at a concentration of about 50 pg/ml to less than 1.micrograms/ml, subcutaneous or submucosal injection or interstitialtissue at a dose in the range of about 1 picograms/ml to about 200.mu.g/ml, local injection at a dose in the range of about picograms/0.1ml to about 4 nanogram/ml to 20 .mu.g/ml, or injection close to majornerve plexus etc. at a dose in the range of about 2 nanograms to 200nanograms/ml as a slow release medication, or injecting a solution of0.01-0.5 ml or more in a physiologic PH adjusted to 7-7.5 PH andosmolarity of 280-300 mOsm.

In one embodiment, a formulation of Wnt or Rock inhibitors is usedcomprising local administering or injecting a solution of 0.01-0.5 ml ormore in a physiologic PH adjusted to 7-7.5 PH and osmolarity of 280-300mOsm to a patient after radiation with plaque therapy. In oneembodiment, a formulation of Wnt or Rock inhibitors is used as acomposition consisting essentially of Rock inhibitors, such as Botox, ina pharmaceutically acceptable formulation and in an amount effective toenhance post-surgical recovery to treat vascular occlusion in the areain the patient wherein the composition is administered at aconcentration up to about 10 micrograms/ml by at least one of slowrelease polycaprolactone, polylactic or polyglycolic acid,microparticles of porous silicone, etc. at a concentration in the rangebetween about 10 picograms/ml to less than 1 micrograms/ml to theradiated area or neuropathy injecting a solution of 0.01-0.5 ml or morein a physiologic PH adjusted to 7-7.5 PH and osmolarity of 280-300 mOsm.

In one embodiment, the composition is administered by injecting asolution of 0.01-0.5 ml or more in a physiologic PH adjusted to 7-7.5 PHand osmolarity of 280-300 mOsm subcutaneously or close to the majortrunk of the irritated nerve or nerve plexus or injected at a dose inthe range of about 1 picograms/ml to about 200 nanograms/ml, at a dosein the range of about 1 picograms/0.1 ml to about 20 nanograms/ml, orretrobulbar injection at a dose in the range of about 20 nanograms/ml toabout 2 micrograms/ml at the site of radiation or chemotherapy inducedneuropathy by injecting in a solution of 0.01-0.5 ml or more in aphysiologic PH adjusted to 7-7.5 PH and osmolarity of 280-300 mOsm.

In one embodiment, the methodology involves administering or injecting asolution of 0.01-0.5 ml or more in a physiologic PH adjusted to 7-7.5 PHand osmolarity of 280-300 mOsm to a patient with a non-toxic dose ofnanoparticle coated Wnt inhibitor and/or Rho Kinase inhibitor havingdiabetic neuropathy, induced neuropathy, and pain induced by ischemiaand neuropathy of the affected area where the neuropathies occur inpatients with long standing diabetes characterized by microvascularabnormalities of the nerve supply, leading to hyper- and hypoaesthesia,burning sensation in the affected area of the long or short nervedistribution, affecting worldwide more than 130 million people where thediagnosis is made by the history of long standing diabetes, poor sugarcontrol, high glycation index, affecting the sensitivity of the skin ofthe foot, etc. is reduced to vibration and pressure and can lead to pinsand needles sensation, or ultimately the vascular occlusion that causesulceration and amputation of the toes or leg, etc., and when cranialnerves are affected it will lead to paresis or paralysis of the fourth,fifth, sixth, and seven nerves associated with diplopia, and theneuropathy can also affect the optic nerve and the sensation of thecornea, leading to dry eye formation and similarly the autonomousnervous system can be affected causing diarrhea, erectile dysfunction,and urinary incontinent, difficulty with swallowing dizzy and possiblefainting.

In one embodiment, in patients with a neuropathy caused by any nerveinjury, diabetes, radiation or chemotherapy or surgery or trauma, thestandard therapy includes blood sugar control and exercise, andadministration of medications, such as an ACE inhibitor to producevasodilatation, antiepileptic medications, including tricyclicantidepressants or anticonvulsants such as pregabalin, valperoic acid,opioids, topical agents, such as Capsaicin, serotonin-neurepinephrinereuptake inhibitors, therapeutic ultrasound, or heating by laser.

In one embodiment one administers topically, or by local injection, asolution of 0.01-0.5 ml or more in a physiologic PH adjusted to 7-7.5 PHand osmolarity of 280-300 mOsm, or oral administration of Rock or Wntinhibitors to prevent vascular damage to the nerve cells, preventendothelial cell loss, provide oxygen to the constricted capillaries,and induce regeneration of the affected nerve axons, prevent secondaryinflammatory processes in diabetes produced by diabetic vasculopathyalong with glycemic control and exercise in patient with neuropathycaused by any nerve injury, diabetes, radiation or chemotherapy orsurgery or trauma.

In one embodiment, in a diabetic patient with diabetic neuropathy, oneadministers topically as an ointment or other preparation or locally byinjection or orally as a preparation of Rock or Wnt inhibitors.

In one embodiment, Wnt inhibitors at a non-toxic dose are used, byinjecting a solution of 0.01-0.5 ml or more in a physiologic PH adjustedto 7-7.5 PH and osmolarity of 280-300 mOsm from the compound such as:FH535, IWP-2, PNU-74654, IWR-1endo. IWR-exo, Demethoxy Curcumin,CCT036477, KY02111, WAY-316606, SFRP, IWP, LGK974, C59, Ant1.4Br/Ant1.4Cl, Ivermectin, Niclosamide, apicularen and bafilomycin, XAV939,XAV939, G007-LK and G244-LM, NSC668036, SB-216763, gemtuzumab etc. smallmolecule Wnt inhibitor PKF118-310, the Wnt/β-catenin pathway inhibitor,and fasudil, a rock inhibitor, Fasudil (HA-1077), a selective RhoA/Rhokinase (ROCK) inhibitor, or Y-27632, small molecule inhibitor of ROCK1and ROCK2, Fasudil1-(5-Isoquinolinesulfonyl)-2 Methylpiperazine CalciumChannel Blockers. Membrane Transport Modulators etc. Canakinumabivermectin, or niclosamide, Botulinum toxine or Botax, all having a goodpenetration into the skin or mucosa or can be delivered as a slowrelease compound implanted or injected inside the affected tissue withany polymeric compound, such as the polymers previously disclosed (e.g.,polycaprolactone, poly(glycolic) acid, poly(lactic) acid, polyanhydride)or lipids that may be formulated as microspheres or dendrimers. As anillustrative example, Fasudil may be mixed with polyvinyl alcohol (PVA),the mixture then dried and coated with ethylene vinyl acetate, thencooled again with PVA. Niclosamide bound with liposomes may be appliedtopically, either in the form of drops or as an aqueous based cream, ormay be administered as an oral preparation or topically as an ointmentor other preparation or locally by injection as nanoparticles and/ormicroparticles with (alpha)-cyclodextrin, or (beta)-cyclodextrin, or(gamma)-cyclodextrin, hydroxypropyl-b-cyclodextrin (bHPCD). In aformulation for topical application, the drug is slowly released overtime as the liposome capsule degrades due to wear and tear from thesurface of the skin or mucosa. In a formulation for intraocularinjection, the liposome capsule degrades due to cellular digestion,other slow release polymers, such as PLA, PGA, Polycaprolactone,microsphere, dendrimers) or as nanoparticles and/or microparticles ofporous silicon or with (alpha)-cyclodextrin, or (beta)-cyclodextrin, or(gamma)-cyclodextrin, hydroxypropyl-b-cyclodextrin (bHPCD) are alsoutilized or some such as Fasudil orally at doses of 40-80 mg. This isequal to 1 microgram/ml to 40 micrograms/ml or to 80 micrograms/ml ormore for topical application having ranges of 40 nanograms/ml to 4micrograms/ml, or 0. 1 micrograms/ml to 40 micrograms/ml or more fortopical applications without having the side effects of steroidpreparation, in addition the following compounds are readily availableand some have been approved by the FDA: potent ROCK inhibitor; orallybioavailable Fasudil hydrochloride, Inhibitor of cyclic nucleotidedependent- and Rho-kinases GSK 269962, Potent and selective ROCKinhibitor GSK 429286, Selective Rho-kinase (ROCK) inhibitor H1152dihydrochloride, Selective Rho-kinase (ROCK) inhibitor Glycyl H 1152dihydrochloride, Selective Rho-kinase (ROCK) inhibitor; more selectiveanalogue of H1152, Cell-permeable, selective Rho-kinase inhibitor OXA 06dihydrochloride, potent ROCK inhibitor PKI1447 dihydrochloride, potentand selective ROCK inhibitor; antitumor SB 772077B, potent Rho-kinaseinhibitor; vasodilator SR 3677 dihydrochloride, potent, selectiveRho-kinase (ROCK) inhibitorTC-57001, potent and highly selective ROCKinhibitor; orally active Y-27632 dihydrochloride, Botox or botulinumtoxin as injectable preparation of topical ointment or with slow releasepolymers described above. Available Wnt inhibitors include smallmolecule Wnt inhibitor PKF118-310, the Wnt/β-catenin pathway inhibitor,niclosamide, ivermectin, etc.

In one embodiment of a patient suffering from diabetic neuropathy orradiation neuropathy, one administers the Wnt and or Rho inhibitorsorally, or injected at a non-toxic dose to the main trunk of theaffected nerve to not only release medication locally, but alsosystemically slowly and be affected for weeks, months or possible years,enhance the circulation to the area, remove the stagnated cytokinereleased in hypoxic tissue and encourage nerve regeneration of thetissue.

In one embodiment, a patient with rheumatoid arthritis, spondylitis,disease affecting small or large joints such as knee or shoulder, wristor fingers or toe, neck or spine vertebrae, nerve compressive effect orinflammatory, bursitis, tendinitis, osteoarthritis, etc., the Wntinhibitors or Rock inhibitors are used by injecting outside or insidethe joint a solution of 0.01-0.5 ml or more in a physiologic PH adjustedto 7-7.5 PH and osmolarity of 280-300 mOsm from the compounds such as:FH535, IWP-2, PNU-74654, IWR-1endo. IWR-exo, Demethoxy Curcumin,CCT036477, KY02111, WAY-316606, SFRP, IWP, LGK974, C59, Ant1.4Br/Ant1.4Cl, Ivermectin, Niclosamide, apicularen and bafilomycin, XAV939,XAV939, G007-LK and G244-LM, NSC668036, SB-216763, gemtuzumab, etc.small molecule Wnt inhibitor PKF118-310, the Wnt/β-catenin pathwayinhibitor and Fasudil, a rock inhibitor Fasudil (HA-1077), a selectiveRhoA/Rho kinase (ROCK) inhibitor, or Y-27632, small molecule inhibitorof ROCK1 and ROCK2, Fasudil1-(5-Isoquinolinesulfonyl)-2 MethylpiperazineCalcium Channel Blockers. Membrane Transport Modulators etc. Canakinumabivermectin, or niclosamide, Botulinum toxin or Botox, all having a goodpenetration into the skin or mucosa or can be delivered as slow releasecompound implanted or injected inside the affected tissue with anypolymeric compound, such as the polymers previously disclosed (e.g.,polycaprolactone, poly(glycolic) acid, poly(lactic) acid, polyanhydride)or lipids that may be formulated as microspheres or dendrimers. As anillustrative example, Fasudil may be mixed with polyvinyl alcohol (PVA),the mixture then dried and coated with ethylene vinyl acetate, thencooled again with PVA. Niclosamide bound with liposomes may be appliedtopically, either in the form of drops or as an aqueous based cream, ormay be administered as an oral preparation or topically as an ointmentor other preparation or locally by injection as nanoparticles and/ormicroparticles with (alpha)-cyclodextrin, or (beta)-cyclodextrin, or(gamma)-cyclodextrin, hydroxypropyl-b-cyclodextrin (bHPCD). In aformulation for topical application, the drug is slowly released overtime as the liposome capsule degrades due to wear and tear from thesurface of the skin or mucosa. In a formulation for intraocularinjection, the liposome capsule degrades due to cellular digestion,other slow release polymers such as PLA, PGA, Polycaprolactone,microsphere, dendrimers) or as nanoparticles and/or microparticles ofporous silicon or with (alpha)-cyclodextrin, or (beta)-cyclodextrin, or(gamma)-cyclodextrin, hydroxypropyl-b-cyclodextrin (bHPCD) are alsoutilized or some such as Fasudil orally at doses of 40-80 mg. This isequal to 1 microgram/ml to 40 micrograms/ml or to 80 micrograms/ml ormore for topical applications having ranges of 40 nanograms/ml to 4micrograms/ml, or 0. 1 micrograms/ml to 40 micrograms/ml or more fortopical applications without having the side effects of steroidpreparation, in addition the following compounds are readily availableand some have been approved by the FDA: potent ROCK inhibitor; orallybioavailable Fasudil hydrochloride, Inhibitor of cyclic nucleotidedependent- and Rho-kinases GSK 269962, Potent and selective ROCKinhibitor GSK 429286, Selective Rho-kinase (ROCK) inhibitor H1152dihydrochloride, Selective Rho-kinase (ROCK) inhibitor Glycyl H 1152dihydrochloride, selective Rho-kinase (ROCK) inhibitor; more selectiveanalogue of H1152, Cell-permeable, selective Rho-kinase inhibitor OXA 06dihydrochloride, potent ROCK inhibitor PKI1447 dihydrochloride, potentand selective ROCK inhibitor; antitumor SB 772077B, potent Rho-kinaseinhibitor; vasodilator SR 3677 dihydrochloride, potent, selectiveRho-kinase (ROCK) inhibitorTC-57001, potent and highly selective ROCKinhibitor; orally active Y-27632 dihydrochloride, Botox or botulinumtoxin as injectable preparation of topical ointment or with slow releasepolymers described above. Available Wnt inhibitors include smallmolecule Wnt inhibitors PKF118-310, the Wnt/β-catenin pathway inhibitor,niclosamide, ivermectin, etc.

In one embodiment, the procedure can be repeated if pain returns.

In one embodiment, the person suffering from fibromyalgia is treatedwith injection of nanoparticles coated with polymeric slow release andthermosensitive compounds, such as chitosan, liposomes or solid lipidnanoparticles, nanoparticles and/or microparticles of porous silicon torelease at a non-toxic dose, NSAIDs, steroid and Rock inhibitors, e.g.,Fasudil, exoenzyme, Y27632, Botox, at picogram to nanogramconcentrations, etc., Wnt inhibitor Niclosamide, etc. at pictogramconcentrations, injected at the site of pain, subcutaneously,intramuscularly, in the neck muscles, back, leg, arm or fore arms'muscles, close to bursae, close to or in the tendon, close to thepainful large or small joints, hips, knees, writs, fingers, toes, closeto the vertebrae, jaw, elbows, etc. or in the joint, at the site ofprior radiation or injury or trauma, while using thermal imaging withthermal energy such as laser, microwave, or preferably with focusedultrasound in a thermal or non-thermal mode at frequencies or 2kilohertz to 1-3 megahertz, thus increasing the temperature of thefunctionalized nanoparticles by a thermal delivery source under thecontrol of the photoacoustic/thermoacoustic imaging system connected toa processor. The energy delivery unit increases the temperature of thefunctionalized nanoparticles from 37° C. to 41° C.-43° C. to melt thetemperature-sensitive coating polymers and release the medicationcombining the thermotherapy with long term release medications thatinhibit inflammatory processes for a long time, and the process can berepeated as needed.

In another embodiment, ultrasound or focused ultrasound is applied withor without a thermal mode to convert the sound energy to electricity,from the injected piezoelectric nanoparticles and to generate aninternal electrical current inducing internalpolarization/depolarization of the cell membranes and induce muscle cellrelaxation.

In another embodiment, after exposure to the focused or non-focusedultrasound waves, applied externally, to the injected antibody coatedpiezoelectric nanoparticles, such as, quartz or graphene, etc.conjugated with drug delivery, inside the tissue where application offocused ultrasound to the nanoparticles produces an electrical pulsethat induces polarization and depolarization of the muscle cells,including the nerve cells for a short period of time at frequencies aslow as 30 Hz-100 kHz for 5-15 second to relax the muscle cells bycreating an internal electrical current from the piezoelectricnanoparticles under ultrasound application.

In another embodiment, there is provided a cancer treatment method usingcontrolled localized thermotherapy. The method comprises the steps of:(i) administering a plurality of nanoparticles to a patient in needthereof so as to target a tumor in the patient, the administerednanoparticles being coated with an antitumor antibody and athermosensitive polymer, and the administered nanoparticles containingquenched fluorescein, liposomes-filled antineoplastic medication, suchas Doxorubicin, fluorescent dextrans, polymer micelles, or nanoparticlescarrying a dye or another dye indicator, such as bubble liposomescontaining air pocket or nanoemulsion of PFC carrying fluorescein,Doxorubicin, a gene and a drug in the thermosensitive polymer coating(e.g., poly(N-isopropylacrylamide); (ii) heating the nanoparticles withan energy source to a temperature of about 41° C. to about 43° C. so asto damage one or more tumor cell membranes at a treatment site of thetumor and melt the thermosensitive polymer coating of the nanoparticles,thereby releasing the fluorescein, Doxorubicin/dye into the circulationof the patient; and (iii) using either focused ultrasound, analternating magnetic field, or laser for accessible lesions ormicrowaves or an alternating magnetic field while measuring thetemperature with photoacoustic imaging with laser or an ultrasoundtransducer variation in the attenuation coefficient, the change inbackscattered energy of the signal (CBE), the backscatteredradio-frequency (RF) echo-shift due to change in the speed of sound andthermal expansion of the medium, and change in the amplitudes of theacoustic harmonics. In these embodiments, an ultrasound thermometrymethod is implemented based on the change in CBE of acoustic harmonics(hCBE), generated by nonlinear propagation of the ultrasound beam in thetissue or a combination thereof to image the temperature, detectlocalized heating and control temperature at that place.

In one embodiment, the antibody-coated gold nanoparticles are filledinside the antibody/aptamer conjugated nanoparticles and medication toenhance the thermal effect of laser where it can be applied byincreasing the temperature in the presence of the gold nanoparticles, orenhancing the effect of radiation by the gold nanoparticles, therebyreducing the need for longer of higher dose or radiation or thermalenergy.

In a further embodiment, the nanoparticles comprise perfluorocarbonliquid (PFCL) nanoparticles, and the method further comprising the stepsof: (iv) additionally heating the nanoparticles with the energy sourceto a temperature of about 56° C. so as to reach the boiling point of thePFCL nanoparticles, thereby creating a detectable cavitation sound; and(v) recording the detectable cavitation sound with an ultrasonicreceiver or microphone so that a control signal is capable being sent tothe energy source to indicate that the heating of target tissue in thepatient is to be stopped, thereby preventing thermal damage tosurrounding healthy cells.

In yet a further embodiment, the antitumor antibody of thenanoparticles/liposomes are in the form of a monoclonal antibody oraptamer.

In still a further embodiment, the thermosensitive polymer coating ofthe pluralities of the nanoparticles further comprises checkpointinhibitors alone or in combination with Rock inhibitors configured to bereleased when the thermosensitive polymer is melted, the checkpointinhibitors being used in a localized immunotherapy treatment proceduretargeting the cells of the tumor without causing damage to the healthycells of the patient, the Rock inhibitor reducing TGF-β production aftertherapy and the subsequent scar formation.

In yet a further embodiment, the energy source heats the nanoparticlesusing focused ultrasonic energy in a thermal mode.

In still a further embodiment, the nanoparticles are selected from agroup consisting of iron oxide gold nanoparticles, gold graphene oxidenanoparticles, gold nanoparticles, silicone nanoparticles, carbonnanoparticles, magnetic nanoparticles, gold nanorods, gold nanoshells,gold nanocages, iron oxide nanotubes, gold nanotubes, carbon nanotubes,and combinations thereof.

In yet a further embodiment, the step of administering a plurality ofnanoparticles to a patient further comprises administering a pluralityof bubble liposomes filled with the dye or indicator or administeringnanoemulsions of perfluorocarbon (PFC) carrying the dye or indicator,the dye or indicator comprising fluorescein, and the liposomes furthercomprising an antineoplastic medication, such as Doxorubicin, etc., tobe released when the local temperature reaches 40-43 degrees C.

In still a further embodiment, the step of heating theantitumor-antibody-coated nanoparticles, liposomes, or micelles releasestumor antigens in the circulation of the patient as a result ofthermally damaging the tumor cells; and the method further comprisingthe steps of: (iv) obtaining from the blood of the patient, the tumorantigens to build a new potent vaccine against many additional tumorspecific antigens, the vaccine combined with antitumor-antibody-coatednanoparticles conjugated with checkpoint inhibitors, and Rock inhibitorsor Wnt inhibitors; (v) administering the vaccine with theantitumor-antibody-coated nanoparticles conjugated with viral-likeparticles (VLP) and/or oncolytic viruses while simultaneously releasingconjugated checkpoint inhibitors, and Rock inhibitors or Wnt inhibitors,from the antitumor-antibody-coated nanoparticles to prevent new or oldtumor cells, metastatic cells, and/or tumor exosomes from beingdisguised from the T-lymphocytes or the patient's natural killer (NK)cells, thereby providing a vaccine for treatment of potentialrecurrences of the same tumor to the patient and enhancing the immuneresponse at the specific location of one or more metastatic lesions,circulating tumor cells, or sessile tumor cells; and (vi) heating theantitumor-antibody-coated nanoparticles and the viral-like particles(VLP) and/or oncolytic viruses of the vaccine so as to kill the VLPand/or oncolytic viruses while leaving the antigenic foreign proteins ofthe VLP and/or oncolytic viruses at the tumor site to enhance an immuneresponse of the patient.

In yet a further embodiment, the cancer treatment method furthercomprises the step of: (iv) systemically administering a monoclonalantibody/aptamer in the form of tocilizumab or appropriate aptamer so asto target interleukin-6 and ornithine phenylacetate, an ammoniascavenger, for the treatment of hepatic encephalopathy in combinationwith low molecular weight heparin reducing inflammatory response in thetissue along with sodium bicarbonate to reduce pH of the inflamedtissue.

In accordance with one or more other embodiments, there is provided acancer treatment method comprising administering to a patient having anearly stage tumor a combination of thermotherapy and immunotherapy,where thermotherapy comprises systemically administeringtumor-antibody/aptamer-coated nanoparticles coated with athermosensitive polymer, cell penetrating peptides (CPPs), and a Wntinhibitor being conjugated with the thermosensitive polymer coating ofthe nanoparticles, the thermotherapy further comprises heating thetumor-antibody-coated nanoparticles using an energy source at the siteof the tumor so as to melt the thermosensitive polymer coating of thenanoparticles, liposomes, and/or micelles and release the Wnt inhibitorto inhibit the Wnt/β-catenin pathway in the cells of the tumor, whereinthe heating of the nanoparticles further damages one or more tumor cellmembranes and releases antigenic material in vivo that activates andstimulates an immunogenic response of the patient at the site of thetumor; and immunotherapy comprises systemically administering thepatient's natural killer (NK) cells/dendritic cells pre-sensitized invitro to the tumor.

In a further embodiment, the method further comprises the step ofremoving cytokines after the immunotherapy by electrophoresis,plasmapheresis, plasma exchange, or ARF6 inhibition or IL-6 inhibitorsso to prevent a cytokine storm, or oral Rho-kinase inhibitors.

In yet a further embodiment, the thermosensitive polymer coating of thenanoparticles further comprises ivermectin or niclosamide configured tobe released when the thermosensitive polymer is melted, the ivermectinor niclosamide inhibiting the Wnt/β-catenin pathway in the cells of thetumor or the nanoparticles of ivernectin, niclosamide or other Wntinhibitors incorporated in liposomes or a combination ofantibody-conjugated liposomes and chitosan-coated nanoparticles for slowrelease of the medication administered locally in the tumor orintra-arterially near the tumor or intravenously to release medicationslowly or with the local increase of the temperature using an energysource.

In still a further embodiment, the thermosensitive polymer coating ofthe nanoparticles further comprises an inhibitory gene(s) and aCRISPR/cas9 complex to stimulate or modify tumor genes at the site ofthe tumor upon release from the thermosensitive polymer coating of thenanoparticles at a temperature of about 41° C. to about 43° C.

In yet a further embodiment, gene modification is done usingantibody-coated nanoparticles conjugated with CRISPR/cas9 mediatedhomology-independent targeted integration (HITI) or homology directedrepair (HDR).

In still a further embodiment, the cancer treatment method furthercomprises the steps of growing a predetermined quantity of tumor cellsof the patient in vitro and applying thermal or non-thermal radiation tothe tumor cells so as to damage or kill the genetic component of thetumor cells, while maintaining the antigenicity of the tumor cellproteins; and administering tumor lysates conjugated withantibody/aptamer coated nanoparticles, VLP, or oncolytic viruses, one ormore Rho-kinase inhibitors, and/or Wnt inhibitors inside the tumor,intra-arterially near the tumor, or intravenously to induce an immuneresponse to the tumor cells, circulating tumor cells, and tumorexosomes.

In accordance with yet one or more other embodiments, there is provideda cancer treatment and imaging method using compressive ultrasoundcomprising the steps of: (i) administering a plurality ofantibody-coated nanoparticles/liposomes to a patient in need thereof soas to target a tumor in the patient, the administered nanoparticlesbeing conjugated with an antitumor antibody and a thermosensitivepolymer, and the nanoparticles administered locally or systemically,containing medication and/or gene and quenched fluorescein in thethermosensitive polymer coating, at least some of the nanoparticlesattaching to surface antigens of tumor cells of the tumor so as to forma tumor cell/nanoparticle complex; (ii) exciting the nanoparticles in afirst compressive non-thermal mode using a ultrasound with a frequencyof generally less than 100 kilohertz (kHz) or less than 300 kHz or 1 MHzand a power of less than one Watt from a power source that generates afocused compressive ultrasonic wave so as to peel off thethermosensitive polymer coating of the nanoparticles by the focusedvibrational force of the ultrasonic wave, temperature by focusedultrasound and imaging it with a transducer and measuring thetemperature by the change in CBE or change in acoustic harmonics (hCBE),generated by nonlinear propagation of the ultrasound beam in the tissueor a combination thereof to image the temperature and controltemperature at that place, thereby releasing fluorescein into thecirculation of the patient and the medication and/or gene at the tumorsite; and (iii) imaging a body region of the patient so as to detect thefluorescein released into the circulation of the patient.

In still a further embodiment, in a second thermal mode, the ultrasonicwave generated by the ultrasound source has a frequency between about150 kilohertz (kHz) and about 300 kilohertz (kHz), and a power of <1Watt/cm² to 50 Watts/cm² or more.

In yet a further embodiment, the nanoparticles comprise antibody oraptamer-coated piezoelectric nanoparticles, and where the focusedcompressive ultrasonic wave in the first compressive non-thermal mode isdelivered in a pulsed manner so as to generate an electric pulse fromthe piezoelectric nanoparticles that depolarizes the one or more tumorcell membranes so as to damage the tumor cells.

In still a further embodiment, antibody-coated piezoelectricnanoparticles are used to generate a sound wave from an electric pulse(from e.g., a battery) from the exposed nanoparticles and a transducer,such as in a telephone receiver, and also the piezoelectricnanoparticles may be exposed to an ultrasonic pulse, which is absorbedby the piezoelectric nanoparticles, thus converting the sound wave intoan electric pulse. Further, these principles may be utilized to image atumor using an external electric pulse and antibody-coated piezoelectricnanoparticles in filled antibody-conjugated liposomes to create a soundwave inside the body in the area to be imaged or apply the externalultrasound transducer to antibody coated piezoelectric nanoparticlesconjugated with a medication to treat a tumor non-invasively inside thebody by depolarizing the tumor cell membrane by the generated electronsfrom the piezoelectric nanoparticles and make the cell membranepermeable to the medication, e.g., anti-cancer medication, etc. by theinternally generated electrical pulses.

In yet a further embodiment, the antibody/aptamer-coated piezoelectricnanoparticles or piezoelectric quantum dots may be administeredintravenously, intra-arterially, topically by injecting submucosally,and/or spraying a nasal mucosa to travel through olfactory nerves to thebrain, antibody/aptamer-conjugated piezoelectric nanoparticles can berendered biocompatible by coating with zirconate titanate,perovskite-based oxides, barium titanate, polyvinylidene fluoride(PVDF), piezoelectric nanoparticles, nanoparticles coated with chitosan,PEG, biotin, streptavidin, radionuclides, cell penetrating peptides(CPPs), activatable cell penetrating peptides (ACCPs), etc. for cellpenetration to deliver drugs or genes, etc.

In accordance with still one or more other embodiments, there isprovided a cancer treatment and imaging method comprising the steps of:(i) systemically administering intravenously antibody-coatedpiezoelectric or pyroelectric nanoparticles to a patient in need thereofso as to target a tumor in the patient, the piezoelectric orpyroelectric nanoparticles being coated with a thermosensitive polymerand/or filled in antibody-conjugated liposomes, and a medication beingconjugated with the thermosensitive polymer coating of the piezoelectricor pyroelectric nanoparticles, the piezoelectric or pyroelectricnanoparticles travel through the body attaching to surface antigens oftumor cells of the tumor so as to form a tumor cell/nanoparticlecomplex; (ii) applying a pulsed electrical current to the piezoelectricor pyroelectric nanoparticles using an electrical source at the site ofthe tumor so as to create an electroacoustic sound from thepiezoelectric or pyroelectric nanoparticles; (iii) recording theelectroacoustic sound generated by the piezoelectric or pyroelectricnanoparticles using a transducer to convert the electroacoustic sound toan electrical signal; and (iv) amplifying and transmitting theelectrical signal to a processor, as is done with an ultrasonic imagingsystem, so that a 1-dimensional, 2-dimensional, or 3-dimensional imageof the tumor structure is able to be generated from the piezoelectricnanoparticles/tumor cells to produce an electroacoustic computedtomogram.

In another embodiment, the electroacoustic system can be used to scanthe entire body, by systemically administering, or intravenouslyadministering, antibody coated piezoelectric or pyroelectricnanoparticles to a patient in need thereof so as to target a tumor inthe patient, a metastatic lesion, circulating tumor cells, and theirexosomes, the piezoelectric or pyroelectric nanoparticles being coatedwith a thermosensitive polymer, and a medication being conjugated withthe thermosensitive polymer coating/CPP of the piezoelectric orpyroelectric nanoparticles, the piezoelectric or pyroelectricnanoparticles travel through the body attaching to surface antigens oftumor cells of the tumor so as to form a tumor cell/nanoparticlecomplex. A pulsed electrical current is applied from moveable electrodesplaced stepwise from the top of the head to the bottom of feet of thepatient to excite the piezoelectric or pyroelectric nanoparticlesattached to the tumor cells while using an electrical pulse at the siteso as to create an electroacoustic sound from the piezoelectric orpyroelectric nanoparticles attached to the tumor cells received by oneor more ultrasound transducers indicating the presence of the tumor inthat area, and reconstructing a full-body electroacoustic tomogram ofthe patient.

In one embodiment, the electrical pulses can vary from 1-30 pulses persecond (30 Hz) up to 1 kHz, or up to 1 MHz or more, or the electricalpulses can be longer than one second to induce various electricalresponses in the tumor to which the antibody coated piezoelectricnanoparticles are attached.

In one embodiment, the lesion discovered by electroacoustic tomogram istreated with focused ultrasound and antibody coated piezoelectricnanoparticles conjugated with either stimulating genes or inhibitorygenes, or genes are replaced using CRISPR cas9 mediatedHomology-Independent Targeted Integration (HITI) or Homology DirectedRepair (HDR).

In another embodiment, the genetic composition of the tumor can bemodified using electroacoustic tomography and administration ofnanoparticle conjugated gene(s) using CRISPR cas9 mediatedHomology-Independent Targeted Integration (HITI) or Homology DirectedRepair (HDR).

In one embodiment, the disease being treated may be a genetic disease,such as Alzheimer's disease, retinal degeneration, Parkinson's disease,muscular dystrophy, diabetes, lung and other genetic diseases of theskin, kidney, or spinal cord, etc.

In a further embodiment, the method further comprises the step of (v)increasing the permeability of one or more tumor cell membranes of thetumor using the pulsed ultrasound, thereby initiating an electric pulsefrom the antibody-conjugated piezoelectric nanoparticles, e.g. quartz orperovskites, coating with zirconate titanate, perovskite-based, oxides,barium titanate, polyvinylidene fluoride (PVDF), piezoelectricnanoparticles, nanoparticles coated with chitosan, PEG, biotin,streptavidin, radionuclides, CPPs, ACCPs to depolarize the tumor cellsattached to the nanoparticles, and minimally facilitating the entry ofthe medication into the depolarized tumor cells of the tumor.

In a further embodiment, the method further comprises the step of (v)increasing the permeability of one or more tumor cell membranes of thetumor using the pulsed focused ultrasound, thereby initiating anelectric pulse from the piezoelectric nanoparticles, e.g., quartz orperovskites, conjugated with a medication, CPP, gene or VLP exposed tothe ultrasound to depolarize the tumor cells, while increasing thetemperature of the tissue with the focused ultrasonic waves andsimultaneously measuring the tissue temperature with a second harmonicwave resulting from back scattered ultrasound generated from the heatedtissue recorded with a transducer located on the patient's skin,connected to an imaging system, recording the tissue temperature andenhancing penetration of the piezoelectric nanoparticles inside the cellthat release the medication, etc. to damage the tumor cells. Thisimaging unit, is in turn, connected via software to the initial focusedultrasound producing unit, controlling the intensity of the pulsedultrasound keeping it at less than 100 kHz intensity and the power atless than 1 W/cm² power to peel off and release medication from thenanoparticles, e.g. from antibody coated quartz or perovskitesnanoparticles conjugated with medication (e.g., similar to the manner inwhich an ultrasonic watch or instrument cleaner removes the dirt andcleaning the watch or instrument) and to simultaneously depolarize thetumor cells attached to the piezoelectric nanoparticles, thusfacilitating the entry of the medication into the depolarized tumor cellmembranes, and as needed, to heat them under the control of power of thefocused ultrasound connected to the thermal imaging system via aprocessor.

In yet a further embodiment, the method further comprises the step of(v) heating the piezoelectric or pyroelectric nanoparticles filled inantibody-coated liposomes using a ultrasound source operating in athermal mode so as to raise the temperature of the tumorcell/nanoparticle complex controllably to a temperature of about 41° C.to about 43° C., thereby damaging one or more tumor cell membranes atthe tumor site and melting the thermosensitive polymer coating orliposomes to release the nanoparticles and the medication at the tumorsite.

In still a further embodiment, in a patient (e.g., a patient with athyroid tumor), the electrical source comprises a battery device with ananode lead positioned on a first side of the body (neck) of the patientand a cathode being located on a second side of the neck of the patient,the pulsed electrical current passing through the neck of the patientfrom the anode to the cathode of the battery device, and where thepulsed electrical current passes through a tumor pretreated withintravenous or intra-arterial injection of the tumor supplying theartery with antibody coated piezoelectric nanoparticles conjugated withmedication attached to the tumor cells, where an electrical pulsecreates an ultrasonic wave from the piezoelectric nanoparticles that canbe recorded by an ultrasonic transducer located on the skin, imaged andlocalize the tumor precisely, then the lesion is treated non-invasivelywith a focused ultrasound beam through the skin and simultaneouslyheating up the tissue to a temperature of 39-40 degrees C. to damage thetumor cells with the thermal energy and depolarizing the tumor cellmembranes by converting the sound waves to an electric pulse todepolarize the tumor cells exposed to the ultrasound, making the cellspermeable to the released medication/gene, etc.

In accordance with still one or more other embodiments, there isprovided a cancer treatment method using focused ultrasound comprisingthe steps of: (i) administering a plurality of piezoelectric orpyroelectric nanoparticles filled in antibody-conjugated liposomes to apatient in need thereof so as to target a tumor in the patient, theadministered piezoelectric or pyroelectric nanoparticles being coatedwith an antitumor antibody and a thermosensitive polymer, and theadministered piezoelectric or pyroelectric nanoparticles containingmedication, a gene, a checkpoint inhibitor, and/or viral-like particles(VLP), 1L-2, ACPP, toxin(s), TLK 7/8 to stimulate a cellular immuneresponse and quenched fluorescein in the thermosensitive polymercoating, at least some of the piezoelectric or pyroelectricnanoparticles attaching to surface antigens of tumor cells of the tumorso as to form a tumor cell/nanoparticle complex; and (ii) stimulatingthe piezoelectric or pyroelectric nanoparticles in a thermal ornon-thermal mode using a ultrasound source that generates a focusedultrasonic wave so as to produce an electrical current from thepiezoelectric or pyroelectric nanoparticles that paralyses cells of thetumor, thus permitting piezoelectric or pyroelectric nanoparticles withthe antitumor antibody coating to enter the cytoplasm of the tumor cellsand release the medication, gene, checkpoint inhibitor, and/or VLP, etc.inside the tumor cells when the medication, gene, checkpoint inhibitor,and/or VLP, CD40 or TLR3, TLR7 is released from the thermosensitivepolymer coating of the piezoelectric or pyroelectric nanoparticles uponthe heating of the nanoparticles to a temperature of about 41° C. toabout 43° C.

In a further embodiment, the antibody coated piezoelectric orpyroelectric nanoparticles are further conjugated with cell penetratingpeptides (CPPs) or activatable cell-penetrating peptides (ACPPs) so toenhance cell penetration into the cells of the tumor prior to treatmentto release the medication inside the tumor cells during non-thermaltherapy with focused ultrasound at pulses of less than 100 kHz and apower of less than 1 Watt/cm².

In yet a further embodiment, the nanoparticles are coated with one ormore antibodies, and the antibody-coated filled liposomes withnanoparticles contain medication and the medication is selected from thegroup consisting of Wnt inhibitors, Rock inhibitors, GSK inhibitors,metformin, buformin, syrosingopine, phenformin, anti-VEGFs, checkpointinhibitors, and combinations thereof further conjugated with cellpenetrating peptides (CPPs) or activatable cell-penetrating peptides(ACPPs) so to enhance cell penetration into the cells of the tumor priorto treatment to release the medication inside the tumor cells duringnon-thermal therapy with focused ultrasound at pulses of less than 100kHz and a power of less than 1 Watt/cm².

In a further embodiment, the administered nanoparticles further containone or more Rock inhibitors in the thermosensitive polymer coating; andthe exciting of the nanoparticles using the non-thermal energy sourcefurther releases the one or more Rock inhibitors from thethermosensitive polymer coating, the one or more Rock inhibitorsreducing TGF-β production after the localized immunotherapy treatmenttherapy, and reducing subsequent scar formation.

In one embodiment, the antibody or aptamer-conjugated liposomes arefilled with polymeric nanoparticles and slow release Rock inhibitors,Wnt inhibitors, GSK-3 inhibitors, anti-integrins, IL-6 and low molecularweight heparin are injected in the tumor tissue, intra-arterially, orintravenously, or administered orally or topically to treat after anyX-ray, proton beam, helium ion radiation or other locally or externallyapplied radiation or any surgery, such as breast or lung etc. or surgeryof the eye, such as refractive surgery, stent implantation incardiovascular surgery, glaucoma, retinal surgery or laser therapy,cosmetic laser therapy, thermal or chemical burns to inhibit release ofTGF-β production and scaring the tissue.

In still a further embodiment, the circulating tumor cells and/or thetumor exosomes are carrying a checkpoint protein that disguises thecirculating tumor cells and/or the tumor exosomes as healthy cells ofthe patient; and the checkpoint inhibitor released from thethermosensitive polymer coating of the nanoparticles blocks thecheckpoint protein of the circulating tumor cells and/or the tumorexosomes so that the circulating tumor cells and/or the tumor exosomesare recognized and killed by the T-cells and killer cells of thepatient.

In yet a further embodiment, when the circulating tumor cells and/or thetumor exosomes are recognized and killed by the T-cells and killer cellsof the patient, tumor antigens are released in the circulation of thepatient; and wherein the method further comprising the steps of: (iii)obtaining from the blood of the patient, the tumor antigens to build anew potent vaccine against many additional tumor specific antigens, thevaccine combined with antitumor-antibody-coated nanoparticles conjugatedwith checkpoint inhibitors, cell penetrating peptides (CPPs), and Rockinhibitors or Wnt inhibitors; and (iv) administering the vaccine withthe antitumor-antibody-coated nanoparticles conjugated with viral-likeparticles (VLP), activatable cell-penetrating peptides (ACPPs), and/oroncolytic viruses while simultaneously releasing conjugated checkpointinhibitors, and Rock inhibitors or Wnt inhibitors, IL-6 inhibitor, etc.from the antitumor-antibody-coated nanoparticles to prevent new or oldtumor cells, metastatic cells, and/or tumor exosomes from beingdisguised from the T-cells and killer cells of the patient, therebyproviding a vaccine for treatment of potential recurrences of the sametumor to the patient and enhancing the immune response at the specificlocation of one or more metastatic lesions, circulating tumor cells, orsessile tumor cells. In one embodiment, the vaccines are injectedsubcutaneously, or preferentially intravenously or intra-arterially orinside a lesion in regular interval of 2-3 months, every 3 months, orevery six months, or every year to eliminate the potentially livingtumors cells in the body and also simultaneously enhance the body'simmune response with interesting benefit for the patient to fight alsopotential infection.

In still a further embodiment, the administered nanoparticles furthercontain oncolytic viruses in the thermosensitive polymer coating; andthe exciting of the nanoparticles using the non-thermal energy sourcefurther releases the oncolytic viruses from the thermosensitive polymercoating, the oncolytic viruses preferentially infecting and killing thetumor cells of the tumor, the circulating tumor cells, and/or the tumorexosomes.

In yet a further embodiment, the non-thermal energy source comprises alow power of about one Watt/cm² low frequency focused ultrasound sourcehaving a frequency between about 40 kilohertz and about 1 megahertz aslow intensity focused ultrasound (LIFU) with thermal response at lowpower of 2-3 W cm².

In another embodiment, a magnetic energy source in the presence ofmagnetic nanoparticles (MNP) generates an alternating magnetic field andheats the MNPs. The temperature values in experimental studies, dependedon two factors, one is AMF current and the other is the concentration ofthe MNP, e.g., at a current of 25 A of AMF, produced 5 degrees C.temperature rise in 20 minutes without MNP in the tissue, whereas aconcentration of just 50 microl of 20 mg/ml MNP increased thetemperature by 5 degrees C. in 10 minutes. Using a software one cancalculate the value of the temperature e.g. at 40-43 is achieved by aknown concentration of MNP and duration of and the current in amps isknown, the system can be also controlled using a PID software.

In yet a further embodiment, the multiple modality, localizedimmunotherapy treatment with the checkpoint inhibitor and the immunestimulator eliminates the need for CAR-T cellular therapy becausethermotherapy damaged tumor cells that release their antigeniccomponents which stimulate activation of cellular immune therapy.

In still a further embodiment, the exciting of the nanoparticles usingthe non-thermal energy source further increases the cell membranepermeability of the tumor cells of the tumor and/or the circulatingtumor cells so as to enhance a penetration of a medication into thetumor cells of the tumor, the circulating tumor cells, and/or tumorexosomes.

In accordance with still one or more other embodiments of the presentinvention, there is provided a cancer treatment method using controlledlocalized thermotherapy, photodynamic therapy, and immunotherapy, themethod comprising the steps of: (i) administering a plurality ofantibody-coated nanoparticles, liposomes, and/or micelles to a patientin need thereof so as to target a tumor in the patient, the administerednanoparticles being coated with an antitumor antibody and athermosensitive polymer conjugated with cell penetrating peptides(CPPs), and the administered nanoparticles containing a photosensitizerand a checkpoint inhibitor and/or an immune stimulator in thethermosensitive polymer coating; and (ii) heating the nanoparticles withan energy source so as to induce a photodynamic effect and melt thethermosensitive polymer coating of the nanoparticles, thereby releasingthe photosensitizer and the checkpoint inhibitor and/or the immunestimulator at a site of the tumor, circulating tumor cells, and/or tumorexosomes so as to provide a multiple modality, localized thermotherapy,photodynamic therapy, and immunotherapy treatment targeting the cells ofthe tumor, the circulating tumor cells, and/or the tumor exosomes byenhancing an immune response of the patient without causing damage tothe healthy cells of the patient, while synergistically preventing thecells of the tumor, the circulating tumor cells, and/or the tumorexosomes from evading the immune system of the patient.

In a further embodiment, the energy source comprises an external orinternal fiber optic device emitting laser light that provides thephotodynamic therapy to the patient by delivering the laser light to thecells of the tumor, the circulating tumor cells, and/or the tumorexosomes to which a photosensitizer has been applied, the photodynamictherapy treating the patient by killing the cells of the tumor, thecirculating tumor cells, and/or the tumor exosomes.

In one embodiment, the pluralities of antibody-coated nanoparticles,liposomes, and/or micelles are conjugated with porphyrin as a cellpenetrating agent, and carry with their thermosensitive polymericcoating one or multiple medications, such as cobimetinib, an MEK1 andMEK2 inhibitor, BRAF and MEK inhibitors, sorafenib, vemurafenib,dabrafenib, pembrolizumab, a check point inhibitor, ipilimumab,nivolumab, olaparib, niraparib, pazopanib, dacarbazine, temozolomide,imatinib, carmustine, cisplatin, carboplatin, and paclitaxel, or acombination thereof for treatment of brain cancers, ovarian cancer,melanoma, lung cancer, in combination with controlled thermotherapyusing an internal or external energy source at temperature of 41-43 C.degree for 1-10 minutes as predetermined by the physician.

In one embodiment, a plurality of liposomes are administered to thepatient, and at least some of the plurality of the liposomes are filledwith nanoparticles coated with a slow release polymer, the slow releasepolymer being selected from the group consisting of polycaprolactone,polylactic acid, and polyglycolic acid, and the nanoparticles disposedin the liposomes contain doxorubicin and porphyrin as a cell penetratingagent (instead of CPP or ACPP), where the porphyrin attaches to the cellmembrane proteins of the tumor cells when released from the liposomesafter being heating to a temperature of about 40° C. to about 43° C. bya laser, LIFU, or AMF, and the doxorubicin entering the tumor cells withease so as to damage the tumor cells.

In one embodiment, the pluralities of antibody coated nanoparticlesincluding piezoelectric nanoparticles or bubble liposomes carryingfluorescein which contain air pockets or perfluorocarbon (PFC)nanoemulsions carry with their thermosensitive polymeric coating one ormultiple antineoplastic medications, check point inhibitors etc. areused for treatment of cancer using a combination of focused ultrasound,or alternating magnetic field (or electromagnetic radiation,non-invasively at a temperature 41-43 degrees C., and then imaged with athermoacoustic imaging system for control of the temperature under thecontrol of a processor controlling thermal energy, along withlow-intensity (1-3 V/cm), low frequency 1-50-kHz or intermediatefrequency 100-200 kHz non-thermal, and 300 kHz to 1 MHz or more tocreate thermal energy, electric fields achieving thermotherapy,electroacoustic imaging using an array of ultrasound transducers,focused ultrasound, or a moving transducer etc. that can move and recordthe produced sounds from the piezoelectric nanoparticles transmitted toa processor or computer to 2-3D electroacoustic images or live video orreconstructed by a computer software, such as in focused ultrasound, anddielectric effect on the cellular components of the tumors, such asbrain tumors, lung cancer, ovarian cancer, breast cancer, prostatecancer, and other cancers, which can be repeated numerous times posttherapy to eliminate the cancer.

In one embodiment, one can diagnose and image an early stage cancer in agenetically predisposed person, using a combination of cancer antibodiesattached to a pluralities of nanoparticles with activatablecell-penetrating peptides (ACPPs), injected systemically intravenouslyor in the cerebrospinal fluid to travel to the suspected tumor locationand attach to the tumor receptors, applying external thermal energy tothe suspected area or body, create a photoacoustic or thermoacousticsound or by the change in CBE or change in acoustic harmonics (hCBE),generated by nonlinear propagation of the ultrasound beam in the tissueor a combination thereof to image the temperature and controltemperature at that place that can be recorded by a transducerindicating thermal expansion of the nanoparticle and the degree of thetemperature imaged, and the location indicating the presence of alesion.

In one embodiment, the antibody-coated pluralities of nanoparticles withactivatable cell-penetrating peptides (ACPPs) are selected frommagnetic, paramagnetic, non-magnetic sphere, rod, nanocarbon, nanowire,nanorod, nanowire, magnetic nanoshells, nanocages, gold nanoshells orsilica-gold nanoshells, silica iron oxide nanoshells, gold coated ferricoxide, quantum dots, magnetic and paramagnetic fullerene, encapsulateferromagnetic nanoclusters, dendrimers, micelles of perfluorocarbonliquids, liposomes/micelles, liposomes in combination with thenanoparticle, nanoshells carrying quenched fluorescein, fluorescentdextrans/dye or Doxorubicin or liposomes or polymer micelles ornanoparticles usually poly(N-isopropyl acrylamide carrying a dyeindicator in thermosensitive polymers that are administered to a patientto seek the potential tumor cells and attach to them exposed to theexternal energy source to release the fluorescein in the circulation at41-43 degrees C. indicating the temperature at the tumor or nanoparticlesite attached to the tumor.

In one embodiment, the energy delivery system is a focused ultrasoundwhich heats up the tissue located at the focal point of the focusedultrasound using a frequency of generally about 2 MHz to 50 MHz or 2 MHzto 100 MHz and a power of more than one Watt and an antibody coatedultrasound contrast agent such as gold nanoparticles (GNPs), goldnanorods (GNRs), gold nanoshells (GNS), graphene oxides (GOs),polypyrrole (PPy) nanocapsules, or nanoshells nanocage magnetic, gold,silicone, carbon, piezoelectric nanotubes, perfluorocarbon (PFC)nanoemulsions, the mixture of magnetic nanoparticles, piezoelectricnanotransducers, boron nitride nanotubes, perovskites, and quenchedfluorescein, Doxorubicin, fluorescent dextrans, or another dye indicatorwith the thermosensitive polymer. The temperature is measurednon-invasively by the estimation of the temperature depending onacoustic harmonic from 26-46 C. degree generated by nonlinear ultrasoundwave.

In one embodiment, only the non-thermal mode, such as generally at afrequency of about 300 kHz and less than one Watt of power, is used withpluralities of antibody coated ultrasound contrast agents, such as goldnanoparticles (GNPs), gold nanorods (GNRs), gold nanoshells (GNS),graphene oxides (GOs), polypyrrole (PPy) nanocapsules, or nanoshells,nanocages; magnetic, gold, silicone, carbon nanotubes, perfluorocarbon(PFC) nanoemulsions, piezoelectric nanotubes, or piezoelectric boronnitride nanotubes, nanogel, liposomes, and/or micelles, and releasesantineoplastic medication/dye, a gene, and a polymeric coatingconjugated with quenched fluorescein or another dye or indicator, suchas polymeric materials, such aspoly(γ-2-(2-(2-methoxyethoxy)-ethoxy)ethoxy-ε-caprolactone)-b-poly(γ-octyloxy-ε-caprolactone)and a dye, have demonstrated significant transition temperatures,allowing improved drug release at low hyperthermia (<40° C.), and therelease is recognized by the presence of the fluorescein in thecirculation in the body or under the nail bed or any other part of thebody.

In one embodiment, the source of the energy is either a laser,ultrasound or an alternating magnetic field and the nanoparticles arerespectively antibody/aptamer conjugated gold, nanoshells or magneticnanoparticles or in combination with antibody/aptamer conjugatedliposomes filled with nanoparticles/polymeric coating/medication thatresponds to thermal energy which is controlled with a software such asPID at temperature of 40-43 C to melt the lipid membrane of theliposomes and/or the polymeric coating of the nanoparticles, and releasethe medication, etc. at the desired site when administered inside alesion or tumor, intra-arterially, or intravenously.

In one embodiment, only the thermal mode of the focused ultrasound isused with pluralities of antibody coated ultrasound contrast agents,such as antibody/aptamer-conjugated gold nanoparticles (GNPs), goldnanorods (GNRs), gold nanoshells (GNS), graphene oxides (GOs),polypyrrole (PPy) nanocapsules, or nanoshells, nanocages; magnetic,gold, silicone, carbon nanotubes, perfluorocarbon (PFC) nanoemulsions,piezoelectric nanotubes, or piezoelectric boron nitride nanotubes,nanogels, liposome and releases an antineoplastic medication/dye, agene, and a polymeric coating conjugated with quenched fluorescein oranother dye or indicator, such as polymeric materials, such aspoly(γ-2-(2-(2-methoxyethoxy)-ethoxy)ethoxy-ε-caprolactone)-b-poly(γ-octyloxy-ε-caprolactone)and a dye, have demonstrated significant transition temperatures,allowing improved drug release at low hyperthermia (40 to 43° C.), andthe release is recognized by the presence of the fluorescein in thecirculation in the body or under the nail bed or any other part of thebody.

In one embodiment, the non-thermal mode and thermal mode of the focusedultrasound is used sequentially with another source of energy radiationor x-ray at lower doses than normally recommended to achieve acomplementary effect on the tumors using pluralities of antibody coatedultrasound contrast agents, such as antibody/aptamer-conjugated goldnanoparticles (GNPs), gold nanorods (GNRs), gold nanoshells (GNS),graphene oxides (GOs), polypyrrole (PPy) nanocapsules, or nanoshells,nanocages; magnetic, gold, silicone, carbon nanotubes, perfluorocarbon(PFC) nanoemulsions, piezoelectric nanotubes, or piezoelectric boronnitride nanotubes, nanogels, liposome and releases antineoplasticmedication/dye, a gene, and a polymeric coating conjugated with quenchedfluorescein another dye or indicator, such as polymeric materials, suchaspoly(γ-2-(2-(2-methoxyethoxy)-ethoxy)ethoxy-ε-caprolactone)-b-poly(γ-octyloxy-ε-caprolactone)and a dye, have demonstrated significant transition temperatures,allowing improved drug release, from antibody coated nanoparticlescarrying Rock inhibitors, Wnt inhibitors to reduce TGF-β productionafter therapy and the subsequent scar formation and antibody coatednanoparticles/checkpoint inhibitors and VLP to prevent the localized orcirculating tumor cells and their exosomes carrying PD-L1 fromdisguising themselves, thus being recognized by the T-cells, whichtogether with killer cells phagocytose them, and at low hyperthermia(<40 to 43° C.), and the release is recognized by the presence of thefluorescein in the circulation in the body or under the nail bed or anyother part of the body.

In one embodiment, the energy delivery system is a focused ultrasound ina compressive non-thermal mode (e.g., at 1-100 kHz), which is visualizedin the tissue by ultrasonic imaging only, the top of the cone-shapedimage of the tissue is seen where the tip of the focused cone exerts thecompressive or pressure force without producing significant thermaleffect, or in combination with another imaging modality such as MRI orCT-scan or PET-scan that defines the focal point of the focusedcompressive ultrasound in relationship with the other body's structures,using an antibody coated ultrasound contrast agent such asantibody/aptamer-conjugated gold nanoparticles (GNPs), gold nanorods(GNRs), gold nanoshells (GNS), graphene oxides (GOs), polypyrrole (PPy)nanocapsules, or nanoshells nanocages, magnetic, gold, silicone, carbonnanotubes, piezoelectric nanotubes, the mixture of magneticnanoparticles, perfluorocarbon (PFC) nanoemulsions, piezoelectricnanotransducers, boron nitride nanotubes, and quenched fluorescein,fluorescent dextrans or another dye or indicator with chitosan,liposomes, or polymer micelles or nanoparticles usually poly(N-isopropylacrylamide carrying a dye, or bubble liposomes carrying fluoresceinwhich contain air pockets or nanoemulsions of PFC measuring the releaseof medication non-invasively by the release of fluorescein in thecirculation.

In one embodiment, the energy delivery system is a focused ultrasound ina thermal mode which is visualized in the tissue by ultrasonic imagingonly by seeing the top of the cone shaped image of the tissue where thetip of the focused cone produces a thermal effect seen as an increase inthermal coagulation and whitening of the tissue effect or in combinationwith another imaging modality such as MRI that defines the focal pointof the focused compressive or pressure mode ultrasound (e.g., at 1-100kHz) in relationship with the other body's structures, using an antibodycoated ultrasound contrast agent, such as antibody/aptamer-conjugatedgold nanoparticles (GNPs), magnetic or paramagnetic nanoparticles, gold,silicone, carbon nanotubes perfluorocarbon (PFC) nanoemulsions,piezoelectric particles or nanotubes, the mixture of magneticnanoparticles, piezoelectric nanotransducers, boron nitride nanotubes,and quenched fluorescein, fluorescent dextrans or another dye orindicator with chitosan, liposomes, or polymer micelles or nanoparticlesusually poly(N-isopropyl acrylamide) carrying a dye or polymericmaterials, such aspoly(γ-2-(2-(2-methoxyethoxy)-ethoxy)ethoxy-ε-caprolactone)-b-poly(γ-octyloxy-ε-caprolactone)7,which have demonstrated significant transition temperatures, allowingimproved drug release at low hyperthermia (e.g., at 40° C.), goldnanorods (GNRs), gold nanoshells (GNS), graphene oxides (GOs),polypyrrole (PPy) nanocapsules, or nanoshells, nanocages, withperfluorocarbon hexane, sealed nanotubes with a non-toxic gas such asSF6, that expands with thermal energy of either focused thermalultrasound, or high intensity focused ultrasound (HIFU), or lowintensity focused ultrasound (LIFU), electromagnetic radiation, RF,microwave or an alternating magnetic field in case of magnetic nanotubesetc., measuring the release of medication non-invasively at temperatureof 41-43 degrees C. seen by the release of fluorescein in thecirculation.

In one embodiment, the patient is exposed to external or internalthermal energy to heat the antibody coated nanoparticles using laser,electromagnetic radiation, visible or infrared light, microwave,radiofrequency, a focused ultrasound, or an alternating magnetic fieldto heat and damage the tumor cells and increase tumor biomarker in thecirculation after the thermotherapy having an important diagnostic value(i.e. indicating presence of a tumor by increased biomarkers) andtherapeutic value for vaccine production after harvesting thecirculating biomarkers and vaccine production for the future managementof the tumor recurrences in the patient to reactivate the immuneresponse and kill the tumor cells.

In one embodiment, the energy source heats the antibody-coatednanoparticles conjugated with medication, etc. using a focused low power.compressive or pressure mode ultrasound, and the nanoparticles withcell penetrating agents or activatable cell penetrating agents andquenched fluorescein or another dye or indicator with polymericmaterials, such aspoly(γ-2-(2-(2-methoxyethoxy)-ethoxy)ethoxy-ε-caprolactone)-b-poly(γ-octyloxy-ε-caprolactone)and a dye have demonstrated significant transition temperatures,allowing improved drug release at low hyperthermia (40° C.), areinjected in the patient's circulation, lymphatic vessels, inside a bodycavity, and the focused ultrasound is applied in the compressive lowpower non-thermal mode 1-40 KHz, where the dye is merely peeled off thenanoparticles or the polymeric carrier by its focused vibrational force.In one embodiment, the energy source, which uses focused compressive orpressure mode ultrasound with low frequencies 1-140 kHz, and <0.3 W isused with antibody coated nanoparticles that are conjugated with amedication, a gene, etc., and contain at least one radioactive agentconjugated with cell penetrating agents or activatable cell penetratingagents and quenched fluorescein, fluorescent dextrans, liposomes filledwith fluorescein or another dye or indicator, or bubble liposomescarrying fluorescein which contain air pockets or nanoemulsions of PFC.The nanoparticles are injected in the patient's circulation, lymphaticvessels, inside a body cavity, or cerebrospinal fluid, and the focusedlow power ultrasound is applied in the compressive or pressure mode(i.e., a non-thermal mode) where the dye is merely peeled off thenanoparticles by the focused vibrational force of the ultrasound whilethe tissue is imaged by the ultrasound or computer-assisted ultrasoundimaging.

In one embodiment, the antibody-coated nanoparticles are injected in thebody, and are exposed to thermal energy, such as electromagneticradiation. The magnetic nanoparticles absorb the energy and expandproducing a sound wave that is known as photoacoustic sound which isrecorded by a transducer and converted to an electrical signaltransmitted to a processor, and then converted to 1D, 2D, or 3Dcomputerized images.

In one embodiment, antibody coated piezoelectric nanoparticlespyroelectric nanoparticles, or perfluorocarbon (PFC) nanoemulsions areinjected inside the body, and the nanoparticles attach to the surfaceantigen of the normal cells or tumor cells and, when exposed to a pulseof electrical current, a sound is created by piezoelectric nanoparticlesinside the body (call an electroacoustic sound) that can be recordedwith a transducer, and then the signal is amplified and forwarded to aprocessor in order to be converted to a 1D, 2D, or 3D image as anelectroacoustic computed tomogram of the structure using a multi-viewHilbert transformation method to recover the unipolar initial pressurefor full-ring electroacoustic computed tomography.

In one embodiment, thermal and electrical energy is used to influencethe permeability of the tumor cell membrane so as to utilize the effector thermotherapy to enhance the effect of chemotherapy or immunetherapy, gene therapy, gene modification using CRISPR-cas9 in benign andmalignant cells, such as brain and spinal cord tumors, breast cancer,lung cancer, prostate and ovarian cancer and melanoma, glioblastoma,retinoblastoma, meduloblastoma, gastrointestinal and genitourinary tumorof sarcoma, etc. Nanoparticles are used having pyroelectric orpiezoelectric characteristics, and the antibody coated nanoparticle tubecomplex is imaged with an electroacoustic computed tomography imagingsystem.

In one embodiment, the antibody coated pluralities of gold,piezoelectric, iron oxide, nanocage, nanotube, nanoshell, magnetic,paramagnetic, pyroelectric, perfluorocarbon (PFC) nanoemulsions, andpiezoelectric nanoparticles are coated with thermosensitive polymers,such as chitosan, liposomes, or polymer micelles or nanoparticlesusually poly(N-isopropyl acrylamide) carrying a dye or bubble liposomescarrying fluorescein which contain air pockets or nanoemulsions of PFCand conjugated with a medication and/or gene, with cell penetratingagents or activatable cell penetrating agents, and with quenchedfluorescein or another dye or indicator, then injected in the patient'scirculation, lymphatic vessels, inside a body cavity, cerebrospinalfluid etc. When the nanoparticles are exposed to electromagneticradiation, microwaves, radiofrequency, focused ultrasound, or analternating magnetic field the temperature of the piezoelectric orpyroelectric nanoparticles and the tumor cell complex is raised to 41-43degrees C. to release the quenched fluorescein or another dye orindicator. When injected into the body, the piezoelectric orpyroelectric nanoparticles attach to the surface antigens of normalcells or the surface antigens of tumor cells and, when exposed to apulse of electrical current with an adjustable signal frequency andvoltage, an acoustic response is produced by electrical stimulation ofthe piezoelectric or pyroelectric nanoparticles inside the body (i.e.,called an electroacoustic sound) that can be recorded by an array oftransducers or a single moving transducer, and then the signal isamplified and forwarded to a processor in order to be converted to a 1D,2D, or 3D or live real time video of a stable lesion or pulsating lesionas an electroacoustic computed tomogram using the movement of thepiezoelectric nanoparticles to be evaluated by the doppler of the bloodvessels for blood flow measurement.

In one embodiment, the energy source vibrates the pluralities ofantibody/medication coated ferroelectric nanoparticles, piezoelectricnanotubes, or pyroelectric nanoparticles conjugated with a medicationand/or gene, and quenched fluorescein, liposomes filled with fluoresceinor polymer micelles or nanoparticles usually poly(N-isopropylacrylamide) carrying another dye or indicator etc., or bubble liposomescarrying fluorescein which contain air pockets or nanoemulsions of PFCusing pulses of electrical current with an adjustable signal frequencyand voltage that create an acoustic response by the electricallystimulated ferroelectric nanoparticles, piezoelectric nanotubes, orpyroelectric nanoparticles inside the body, which are recorded by one ormultiple transducers located in different parts of the body. The signalis then amplified and forwarded to a processor in order to be convertedto a 1D, 2D, or 3D image by electroacoustic computed tomography. Thelesion or lesions to which the piezoelectric nanoparticles and othernanoparticles are attached are recognized by electroacoustic computedtomography, and are radiated with pulses of non-thermal focusedultrasound low intensity focused ultrasound (LIFU), thereby releasingthe dye, medication, gene etc. by vibrational force of ultrasound in anon-thermal mode under observation of the electroacoustic imaging, notonly indicating the presence of tumor cells using universalback-projection (UBP) and time reversal algorithm, etc., but also thelesion is treated simultaneously with the medication/gene and imagedboth by the focused ultrasound and electroacoustic imaging. Thepiezoelectric or pyroelectric nanoparticle, or nanotube/cell complex,which releases the medication, is verified by the release of fluoresceinin the circulation.

In one embodiment, the non-thermal focused high power ultrasound energysource vibrates the antibody/medication coated magnetic nanoparticlesconjugated with a medication and/or gene, and quenched fluorescein oranother dye or indicator, etc.

In one embodiment, using an alternating magnetic field andantibody/aptamer-coated magnetic nanoparticles without or with producinga significant thermal response, depending on the concentrations of theMNPs coated with thermosensitive polymers, such as polycaprolactone orincorporated in the liposomes or micelles to break down at temperatureof 40-43 C, thus releasing the dye, medication, gene, etc. byvibrational force or thermal energy created in the magneticnanoparticle, and the release of dye/medication is verified by therelease of the medication locally and the temperature is controlled by asoftware that indicates the temperature created at the site of injectionof MNP predetermined depending on the concentration and time of durationof AMF and its electrical current.

In one embodiment, a robotic arm may be used to heat the tissue or thetumor at the focal point of the focused ultrasound, thus producing a 2to 3 dimensional image of the ultrasound, while the thermal energy iscontrolled automatically by the software based on the ultrasoundtransducer variation in the attenuation coefficient, the change inbackscattered energy of the signal (CBE), the backscatteredradio-frequency (RF) echo-shift due to change in the speed of sound, andthermal expansion of the medium, and change in the amplitudes of theacoustic harmonics. In this embodiment, an ultrasound thermometry methodis implemented based on the change in CBE of acoustic harmonics (hCBE),generated by nonlinear propagation of the ultrasound beam in the tissueor a combination thereof to image the temperature, detect localizedheating and control temperature at that place, energy while the focalpoint is under the observation of the 2-3 D imaging system and heats upthe entire tumor in a 50 micron by 50 micron size focal point, thusreleasing the medication, gene, etc. from the antibody-coatednanoparticles/medication injected in the circulation throughout thetumor.

In one embodiment, non-thermal low power ultrasound (LIFU) is appliedfor 1-5 minutes or more and therapy is combined with MRI, therebyeliminating cavity formation of the high power high-intensity focusedultrasound (HIFU) and potential scar and bubble formation with HIFU inthe path of the ultrasound, and reducing the time of the ultrasoundsurgery with HIFU and its complications.

In one embodiment, the antibody-coated pluralities of gold, iron oxide,silica, nanocage, nanotube, nanoshell, magnetic, paramagnetic,pyroelectric, and piezoelectric nanoparticles are coated withthermosensitive polymers, such as chitosan, liposomes, or polymermicelles or nanoparticles usually poly(N-isopropyl acrylamide) carryinga dye quenched with fluorescein or another dye or indicator etc. andconjugated with a medication and/or gene, and conjugated with Wntinhibitors or Rock inhibitors with cell penetrating agents oractivatable cell penetrating agents and quenched fluorescein injected inthe patient's circulation, lymphatic vessels, inside a body cavity,cerebrospinal fluid, etc. so as to be attached to the surface antigen ofnormal cells or of tumor cells, and to expose the piezoelectricnanoparticles to pulses of electrical current with adjustable signalfrequency and voltage thereby creating an electroacoustic sound from thepiezoelectric nanoparticles that can be recorded with one or multipletransducers. The signal is then amplified and forwarded to a processorso as to be converted to a 1D, 2D, or 3D image or video as anelectroacoustic computed tomogram, using simultaneously focused lowpower ultrasound energy in a thermal mode (LIFU) to raise thetemperature of nanoparticles/tumor cells complex to 41-43 degrees C., ormore thereby releasing the fluorescein and the medication using focusedultrasound energy in the thermal mode under the control of a processorcontrolling the thermal energy intensity and duration of the focusedultrasound, while imaging the lesion with the change in CBE of acousticharmonics (hCBE), generated by nonlinear propagation of the ultrasoundbeam in the tissue or a combination thereof to image the temperature andelectroacoustic computerized tomography or videography.

In one embodiment, the antibody coated pluralities of gold,piezoelectric, iron oxide, nanocage, nanotube, nanoshell, magnetic,paramagnetic, pyroelectric and piezoelectric nanoparticles are coatedwith thermosensitive polymers, such as chitosan, liposomes, or polymermicelles or nanoparticles usually poly(N-isopropyl acrylamide) carryinga dye quenched with fluorescein or another dye or indicator etc. orbubble liposomes carrying fluorescein which contain air pockets ornanoemulsions of PFC conjugated with a medication, and conjugated withcell penetrating agents or activatable cell penetrating agents andquenched fluorescein, and injected in the patient's circulation,lymphatic vessels, inside a body cavity, cerebrospinal fluid, etc. suchthat the dye is released when the nanoparticles are exposed to low powerfocused ultrasound energy in a non-thermal compressive mode (LIFU)1-140kHz and 0.1-1 Watt to vibrate the piezoelectric, pyroelectricnanoparticles/tumor cells complex that are injected inside the body andare attached to the surface antigen of normal cells or of tumor cells,and when exposed to pulses of electrical current with an adjustablesignal frequency and voltage, an electroacoustic sound is created insidethe body from the piezoelectric nanoparticles that can be recorded witha transducer and the change in CBE of acoustic harmonics (hCBE),generated by nonlinear propagation of the ultrasound wave in the tissueor a combination thereof to image the temperature. The signal is thenamplified and forwarded to a processor to be converted to a 1D, 2D, or3D image as an electroacoustic computed tomogram and ultrasound image ofthe structure, and the dye/medication is released under observation ofthe lesion as video-electroacoustic imaging.

In one embodiment, antibody/medication coated gold, iron oxide,nanocage, nanotube, nanoshell, magnetic, paramagnetic, pyroelectric andpiezoelectric nanoparticles are conjugated with a medication andcheckpoint inhibitors and immune stimulators such as VLP, TLR3, TLR7,TLR8 or CD40 and quenched fluorescein or another dye or indicator, etc.so that the medications are released using high power focused ultrasoundenergy in a thermal mode alternating with focused ultrasound in anon-thermal compressive mode at 1-140 kHz to one MHz or more, and lessthan 1 Watt power (LIFU) while antibody coated nanoparticles/checkpointinhibitors and VLP attach to the localized or circulating tumor cellsand their exosomes, which are carrying a checkpoint protein, such asPD-L1 (to disguise themselves), and the tumor cells are recognized bythe T-cells, which together with killer cells phagocytose them, andenhance the immune response to the tumor and its exosomes and thecirculating cells. The T-cells destroy the tumor cells, circulatingtumor cells, and their exosomes as a result of the VLPs and checkpointinhibitors that are attached to the tumor cells, circulating tumorcells, and their exosomes. The VLPs make the tumor cells, circulatingtumor cells, and their exosomes visible to the T-cells, which destroythem.

In one embodiment, the energy source heats the antibody/medicationcoated pyroelectric or piezoelectric nanoparticles conjugated withmedication and checkpoint inhibitors, VLP, etc. and quenched fluoresceinor another dye or indicator, etc. to be released at temperature of 41-43degrees C. for the desired time using focused ultrasonic energy in athermal mode under the control of a processor controlling the intensityof the focused ultrasound for a desired duration, while antibody coatednanoparticles/checkpoint inhibitors and VLP, TLR3, TLR7, TLR8 or CD40attach to the localized or circulating tumor cells and their exosomes,which are carrying a checkpoint protein, such as PD-L1 (to disguisethemselves), and the tumor cells are recognized by the T-cells, whichtogether with killer cells phagocytose them, and enhance the immuneresponse to the tumor and its exosomes and the circulating cells.

In one embodiment, the focused low power focused ultrasound energysource heats the antibody/medication coated gold, iron oxide, nanocage,nanotube, nanoshell, or piezoelectric nanoparticles using focusedultrasound so as to heat the tumor cell nanoparticle complex conjugatedwith quenched fluorescein or another dye or indicator etc., and VLP,CD40 or TLR3, TLR7 and a pulse of electric current is applied to inducea sound from the piezoelectric nanoparticles, image the lesion and torelease fluorescein and medication at 41-43 degrees C., and the changein CBE of acoustic harmonics (hCBE), generated by nonlinear propagationof the ultrasound beam in the tissue or a combination thereof to imagethe temperature while maintaining the intensity, duration of theultrasound energy or reducing its intensity to a desired level, and fora desired time by a software obtaining blood samples to evaluateincreased biomarkers in the circulation and produce a vaccine withviral-like particles (VLPs), or oncolytic viruses, T-Vec andadministering them with thermotherapy and releasing them at thetemperature or 41-43 degrees C. that also damages or kills the VLP andoncolytic viruses such as T-Vec while leaving their antigenic foreignproteins in the tumor site to enhance an immune response, whilereleasing checkpoint inhibitors, such as PD-1, PD-11, CTLA-4, Jagged 1inhibitor 15D11, while antibody coated nanoparticles/checkpointinhibitors and VLP, CD40 or TLR3, TLR7 attach to the localized orcirculating tumor cells and their exosomes, which are carrying acheckpoint protein, such as PD-L1 (to disguise themselves), and thetumor cells are recognized by the T-cells, which together with killercells phagocytose them, and enhance the immune response to the tumor andits exosomes and the circulating cells, etc. and anti-VEGF Rockinhibitors, such as Fasudil, or Wnt inhibitors, such as niclosamide forfuture use, as needed (e.g. every six months or once a year) reducingTGF-β production after therapy and the subsequent scar formation.

In one embodiment, antibody coated iron oxide, nanocage, nanotube, nanoshell, magnetic, paramagnetic, pyroelectric and piezoelectricnanoparticles are conjugated with medication, checkpoint inhibitors, andquenched fluorescein or bubble liposomes carrying fluorescein whichcontain air pockets or nanoemulsions of PFC to be released using focusedultrasonic in a compressive mode at 1-40 kHz where the medications arefrom the group consisting of Wnt inhibitors, rock inhibitors, metformin,anti-VEGFs, Doxorubicin, cyclophosphamide, antibiotics,anti-inflammatory, mammalian target of rapamycin, etc.

In one embodiment, glucose metabolism activates the Ras signaling thatregulates the cell proliferation, along or independently from the Wntactivation and glucose triggers activation of MEK and ERK and inhibitionof glucose metabolism at the tumor cell level with metformin, etc.delivered with antibody coated nanoparticles of iron oxide, nanocage,nanotube, nanoshell, magnetic, paramagnetic, pyroelectric . nanobubbles,or microbubbles and piezoelectric nanoparticles conjugated with amedication selected from the group consisting of Wnt inhibitors, Rockinhibitors, metformin, buformin, and syrosingopine, phenformin,anti-VEGFs, and checkpoint inhibitors, VLP, IL-2, or bee toxins, etc.,enzymes such as Matrix metalloproteinases as immune stimulators andquenched fluorescein or another dye or indicator, etc. are releasedusing focused ultrasonic energy in a compressive mode at 20-50-kHz wherethe medications/dye are released or LIFU at temperatures of 40-43 Caffecting the membranes of the cancer cells and their metabolism moresignificantly than the normal surrounding normal cells and release ofVLP, CD40 or TLR3, TLR7 etc. as stimulators and checkpoint inhibitorsenhance the immune response to the tumor locally and also theirinvisible metastatic lesions, circulating tumor cells and their exosomescarrying the same tumor receptors and PD-L1 on their membranes.

In one embodiment, antibody/medication coated gold, iron oxide,nanocage, nanotube, nanoshell, magnetic, paramagnetic, pyroelectric, andpiezoelectric nanoparticles are conjugated with medication andcheckpoint inhibitors, VLP, or immune stimulators and quenchedfluorescein or another dye or indicator, etc. to be released using lowintensity focused ultrasound LIFU in a thermal mode alternating withfocused compressive ultrasound in a non-thermal mode.

In one embodiment, the antibody/medication piezoelectric nanoparticles,pyroelectric, or perovskites nanoparticles are re-injected to reveal theexistent of a lesion or tumor after the tumor has been treatedpreviously under pulses of electrical current with an adjustable signalfrequency and voltage that produces an electroacoustic sound wave fromthe piezoelectric nanoparticles or pyroelectric or perovskites that canbe captured, amplified, and converted to a signal producing an image oras an electroacoustic computed tomogram indicating either persistence ofthe tumor cells, recurrences, or metastatic lesion or the tumor whileelectric pulses of 30-100 Hz and more paralyses the tumor cells bydepolarizing the membrane potential and making their cell membraneaccessible to medication.

In one embodiment, the antibody-coated pluralities of gold, iron oxide,silica, nanocage, nanotube, nanoshell, magnetic, paramagnetic,pyroelectric and piezoelectric nanoparticles are coated withthermosensitive polymers, such as chitosan, liposomes, or polymermicelles or nanoparticles usually poly(N-isopropyl acrylamide) carry adye quenched with fluorescein or another dye or indicator, etc.conjugated with a medication and/or gene, and conjugated with Wntinhibitors or rock inhibitors and with cell penetrating agents oractivatable cell penetrating agents and quenched fluorescein or anotherdye or indicator etc. injected in the patient's circulation, lymphaticvessels, inside a body cavity, cerebrospinal fluid, etc. so as to beattached to the surface antigen of normal cells or of tumor cells, andthe piezoelectric nanoparticles are exposed to a pulse of electricalcurrent, thereby creating an electroacoustic sound from thepiezoelectric nanoparticles that can be recorded with a transducer. Thetransducer signal is then amplified and forwarded to a processor so asto be converted to a 1D, 2D, or 3D image using focused low powerultrasound energy in a non-thermal mode, thus releasing the fluoresceinand the medication and/or gene etc. using the focused non-thermal modeto strip away the dye, medications, and/or gene under the control of aprocessor controlling the thermal ultrasonic intensity and duration ofthe ultrasound, while imaging the lesion with the ultrasound along withelectroacoustic 2-D or 3-D images as an electroacoustic computedtomogram or in combination with another imaging modality such as MRI orCT-scan or PET-scan that defines the focal point of the focusedcompressive ultrasound in relationship with the other body's structures.

In one embodiment, iron oxide, nanocage, nanotube, nanoshell, magnetic,paramagnetic, pyroelectric and piezoelectric antibody/medication coatednanoparticles contain at least one radioactive agent conjugated withmedication and checkpoint inhibitors Botulinum toxin, VLP, etc. Rockinhibitors, such as Fasudil, netarsudil, and quenched fluorescein oranother dye or indicator, etc. so as to be released using focusedultrasonic in thermal mode alternating with a non-thermal focusedcompressive low power ultrasound mode and the repeated ultrasound pulsesgenerate an electric pulse from the piezoelectric nanoparticles thatdamages the tumor cell by depolarizing their cell membrane whileantibody coated nanoparticles/checkpoint inhibitors and VLP attach tothe localized or circulating tumor cells and their exosomes, which arecarrying a checkpoint protein, such as PD-L1 (to disguise themselves),and the tumor cells are recognized by the T-cells, which together withkiller cells phagocytose them, and enhance the immune response to thetumor and its exosomes and the circulating cells.

In one embodiment, iron oxide, nanocage, nanotube, nanoshell, magnetic,paramagnetic, pyroelectric, and/or piezoelectric antibody coatednanoparticles are conjugated with a rock inhibitor, antineoplasticmedication, RNAi, siRNA, and checkpoint inhibitors, Rock inhibitors,such as Fasudil, etc., and quenched fluorescein or another dye orindicator, etc., and are exposed to pulses of electrical current with anadjustable signal frequency and voltage so that an electroacoustic soundis created inside the body from the piezoelectric nanoparticles that canbe recorded with a transducer. The transducer signal is then amplifiedand forwarded to a processor in order to be converted to a 1D, 2D, or 3Dimage as an electroacoustic computed tomogram, and to release themedication using focused Low power ultrasound in a thermal mode LIFU(e.g., at a frequency of 50-1 MHz) alternating with repeated low powerfocused ultrasound pulses generate an electric pulse from theantibody/aptamer-conjugated piezoelectric nanoparticles that damages thetumor cell by depolarizing their cell membrane.

In one embodiment, pluralities of iron oxide, gold, nanocage, nanotube,nanoshell, magnetic, paramagnetic, pyroelectric, and piezoelectricantibody coated nanoparticles are conjugated with rock inhibitors, anantineoplastic medication, genes, CRISPR-cas9, checkpoint inhibitors,Rock inhibitors such as Fasudil, etc. and quenched fluorescein oranother dye or indicator, etc., and then exposed to pulses of electricalcurrent with an adjustable signal frequency and voltage so that aelectroacoustic sound is created inside the body from the piezoelectricnanoparticles that can be recorded with a transducer. The transducersignal is amplified and forwarded to a processor so as to be convertedto a 1D, 2D, or 3D image as an electroacoustic computed tomogram, andthe gene, CRISPR-cas9, or medication is alone or in combination,released using focused low power compressive ultrasound to strip thecoatings off the nanoparticles by vibrational forces of the non-thermalmode of ultrasound and to modify the gene using CRISPR-cas9 mediatedHomology-Independent Targeted Integration (HITI) or Homology DirectedRepair (HDR).

In one embodiment, iron oxide, nanocage, nanotube, nanoshell, magnetic,paramagnetic, pyroelectric, and piezoelectric antibody coatednanoparticles conjugated with a rock inhibitor, antineoplasticmedication, CRISPR-cas9, a corrective gene, and/or quenched fluoresceinor another dye or indicator, etc. are exposed to pulses of electricalcurrent with an adjustable signal frequency and voltage so that anelectroacoustic sound is created inside the body from the piezoelectricnanoparticles that can be recorded with a transducer. The transducersignal is then amplified and forwarded to a processor to be converted toa 1D, 2D, or 3D image as an electroacoustic computed tomogram, and thenanoparticle coating is released using focused low power ultrasound in acompressive ultrasound mode so as to modify the gene using CRISPR-cas9mediated Homology-Independent Targeted Integration (HITI) or HomologyDirected Repair (HDR) while imaging the lesion.

In one embodiment, the transducer of a focused low power, compressive,or thermal high power ultrasound is positioned on the surface of thebody to aim the focal point of the ultrasound at specific part of thelesion now made visible by electroacoustic imaging technology, such asits center or the borders of the lesion, or the focal point can move sothat the entire lesion is treated with low power,compressive or lowthermal focused ultrasound providing thermal energy to heatantibody/medication coated nanoparticles and its thermosensitivepolymers carrying a dye or gene, checkpoint inhibitors, such as Fasudil,and its derivatives etc., VLP or oncolytic viruses, such as T-Vec,monoclonal antibodies, IL-2, bee toxins, immune stimulators along withcheckpoint inhibitors, medication are released and damage already heatedtumor cells at a temperature of 41-43 degrees C. and release dye andmedication from the nanoparticles indicating the temperature of 41-43degrees C. has been achieved which can be controlled by the processor orcomputer controlling energy delivery, under direct imaging the tumor,orthe lesion or modify the genetic component of the tissue withappropriate genes and CRISPR Cas 9 mediated Homology-IndependentTargeted Integration (HITI) or Homology Directed Repair (HDR) or enhanceimmune therapy by the dead VLP or oncolytic viruses, such as T-Vec andrelease other immune stimulators and checkpoint inhibitors to enhancethe immune response to the tumor locally and also their invisiblemetastatic lesions and initiate an immune response to the tumor cells,circulating cells and their circulating exosomes and eliminate them.

In one embodiment, the antibody coated nanoparticles, nanospheres,liposomes, nanowires, nanorods, nanocages, nanoshells, magnetic,ferromagnetic, piezoelectric, pyroelectric, nanotubes, zinc oxide,barium titanate, iron oxide, gold, gold silica, strontium titanate,perovskites, and polyvinylidene fluoride, boron nitride nanotubes or acombination thereof are coated with a thermosensitive polymer, such aschitosan, polysaccharides, (e.g., glycol chitosan, poly-L-Lysine (PLL),polyethylene imine (PEI), polylactic, polyglycolic, polyaspartic acid orcopolymers), a cationic polymer, such as polylysine and polyethyleneimine, liposomes, or polymer micelles or nanoparticles usuallypoly(N-isopropyl acrylamide) carrying a dye and conjugated with a rockinhibitor, checkpoint inhibitors and immune stimulators such as VLP,IL-2, toxine, MMPases, to initiate an immune response, CRISPR-cas9 and agene with cell penetrating agents or activatable cell penetrating agentsand quenched fluorescein or another dye or indicator, etc. to bereleased using focused non-thermal ultrasound (LIFU) in a compressivemode to modify the defective gene using CRISPR-cas9 mediatedHomology-Independent Targeted Integration (HITI) or Homology DirectedRepair (HDR) under observation by an electroacoustic computed tomogramand) and induce an immune response by released of VLP, etc. asstimulators and checkpoint inhibitors enhance the immune response to thetumor locally and also their invisible metastatic lesions elsewhere andinitiate an immune response to the tumor cells, circulating cells, andtheir exosomes and eliminate them.

In one embodiment, the pluralities of antibody coated nanoparticles,nanospheres, liposomes, nanowires, nanorods, nanocages, nanoshells,nanotubes; magnetic, ferromagnetic, piezoelectric, pyroelectricnanotubes; zinc oxide, barium titanate, iron oxide, gold, gold silica,strontium titanate, perovskites, and polyvinylidene fluoride boronnitride nanotubes or a combination thereof are coated with athermosensitive polymer, such as chitosan, polysaccharides, e.g., glycolchitosan, poly-L-Lysine (PLL), polyethylene imine (PEI), polylactic,polyglycolic, polyaspartic acid or copolymers, a cationic polymer suchas polylysine and polyethylene imine, liposomes, or polymer micelles ornanoparticles usually poly(N-isopropyl acrylamide) carrying a dyeconjugated with a rock inhibitor, antineoplastic medication, CRISPR-cas9and VLP or other immune stimulators, oncolytic viruses, such as T-Vec,monoclonal antibodies, IL-2, bee toxins or other immune stimulators,checkpoint inhibitors, and a gene with cell penetrating agents oractivatable cell penetrating agents and quenched fluorescein or anotherdye or indicator/medication etc. to be released using focused ultrasoundin a non-thermal compressive or pressure mode along with pulses ofelectrical current with an adjustable signal frequency and voltageinducing a sound wave in the piezoelectric nanoparticles that can beverified by a transducer attached to the patient's body confirming thelocation of the tumor cells attached to the antibody coatedpiezoelectric nanoparticles as an electroacoustic computed tomogram tomodify the defective gene using CRISPR cas9 mediatedHomology-Independent Targeted Integration (HITI) or Homology DirectedRepair (HDR), or to release antineoplastic medication at that place orcheckpoint inhibitors, VLP, Rock inhibitors, such as Fasudil, etc., Wntinhibitors discovering a single tumor or many metastatic cells locatedadjacent to the original lesion, in the lymph nodes, or in thecirculation under observation by an electroacoustic computed tomogramand inducing an immune response by the release of VLP, etc. asstimulators and checkpoint inhibitors enhancing the immune response tothe tumor locally and also their invisible metastatic lesions elsewhereand initiate an immune response to the tumor cells, circulating cellsand their exosomes.

In one embodiment, the pluralities of antibody/medication coatednanoparticles, nanospheres, liposomes, nanowires, nanorods, nanocages,nanoshells, magnetic, non-magnetic, paramagnetic, pyroelectricpiezoelectric nanotubes; magnetic, ferromagnetic, piezoelectric,pyroelectric nanotubes; zinc oxide quantum dots, quartz, bariumtitanate, iron oxide, gold, gold silica, strontium titanate,perovskites, and polyvinylidene fluoride boron nitride nanotubes;ferroelectric, pyroelectric or piezoelectric materials enclosed by apolymeric membrane and containing a drug, a gene, and/or a combinationthereof are coated with a polymer, such as chitosan, polysaccharides,e.g., glycol, chitosan, poly-L-Lysine (PLL), polyethylene imine (PEI),polylactic, polyglycolic, polyaspartic acid or copolymers, cationicpolymer, such as polylysine and polyethyleneimine, polyethylene glycol,biotin, streptavidin are injected with cell penetrating agents oractivatable cell penetrating agents and quenched fluorescein orliposomes, or polymer micelles or nanoparticles usually poly(N-isopropylacrylamide) carrying fluorescein dye or another dye orindicator/medication, etc. in the patient's circulation lymphaticvessels, inside a body cavity, in cerebrospinal fluid, in the eye, inthe bladder, in the uterus, in the peritoneal cavity, and stimulatedwith pulses of electrical current at predetermined intervals atdifferent places in the body while listening to the production of asound wave which has the same frequency as the frequency of the pulsesof electrical current applied to the tissue indicating the presence of asuspected lesion by the production of a sound wave by the piezoelectricnanoparticle/tumor or exosomes, recording the sound waves by anultrasonic transducer or a microphone, and converting the sound wave toan electrical signal sending it to an imaging oscilloscope, videoscreen, to create 1-dimensional, 2-dimensional, or 3-dimensional imagesas a tomogram using a computer software indicating the presence and theshape of the lesion in the body or its absence using an electroacousticcomputed tomography.

In one embodiment, the abovementioned electroacoustic computedtomography may be made as a tube, half tube, partial tube like MRI, orCT-scan or PET-scan units in which the patient can lay down and thetable moves the patient in or out of the system while multiple rows orelectrical connection is made like an electrocardiogram recordingelectrode, but in this case, they represent anode or cathodes arrays ofthe system so that the entire patient body may be scanned or imagedusing a computer controlled recording, and electroacoustic tomography isdone in a less expensive and time consuming way than is needed for anMRI with its specific room or CT-scan or PET-scan, but without exposingthe patient to radiation.

In one embodiment, the antibody coated pluralities of gold,piezoelectric, iron oxide, nanocage, nanotube, nanoshell, magnetic,paramagnetic, pyroelectric, and/or piezoelectric nanoparticles, etc. arecoated thermosensitive polymers, such as chitosan, quenched withfluorescein or another dye or indicator/medication etc. and conjugatedwith an antineoplastic medication, gene, CRISPR-cas9 conjugated withcell penetrating agents or activatable cell penetrating agents injectedin the patient's circulation, lymphatic vessels, inside a body cavity,cerebrospinal fluid, etc. The functionalized nanoparticles are exposedto electromagnetic radiation, microwaves, or radiofrequency radiation,or focused high power ultrasound 244 (see FIG. 9) in a thermal ornon-thermal low power mode or non-focused ultrasound, or an alternatingmagnetic field and/or electrical current generated by a battery 230 (seeFIG. 9) where low electrical current from a battery 230 passes from oneside of the skin (i.e., the anode 232) through the body 238 and a lesionor tumor 234 to the cathode electrode 236 positioned on the oppositeside of the skin on the body 238 to raise the temperature of thepiezoelectric or pyroelectric nanoparticles that are injected inside thebody 238 to be attached to the surface antigens of the normal cells orof the tumor cells and create a nanoparticle/tumor cell complex heatedto 41-43 degrees C., and when exposed to pulses of electrical currentgenerated by the battery 230 with an adjustable signal frequency andvoltage, an acoustic response is produced by electrical stimulation ofpiezoelectric nanoparticles inside the body 238 that is calledelectroacoustic sounds or signals which can be captured with atransducer (e.g., ultrasound transducer 240 in FIG. 9), or microphone,converted to an electrical signal and is forwarded to a processor to beconverted to a 1D, 2D, or 3D image 42 as an electroacoustic computedtomogram while the electrical pulse generated in the piezoelectricnanoparticles drives the medication, gene, in the tumor cells locally todamage the tumor cells by multiple modes of the therapy appliednon-invasively under observation.

In another embodiment, in a patient with a thyroid tumor 250 (see FIG.10), an electrical source comprises a battery device 246 with an anode248 being positioned on a first side of the body of the patient and acathode 252 being located on a second side of the body of the patient,the pulsed electrical current (as diagrammatically indicated by thecurrent lines in FIG. 10) passing through the body (e.g., the neck) ofthe patient from the anode 248 to the cathode 252 of the battery device246, and where the pulsed electrical current passes through the tumor250 which has been pretreated with antibody coated piezoelectricnanoparticles conjugated with medication, and attached to the tumorcells. In FIG. 10, it can be seen that the thyroid gland 256 of thepatient, which is disposed around the trachea 254, comprises the rightlobe 258, the left lobe 262, and the isthmus 260 connecting the rightand left lobes 258, 262. Thyroid cartilage 264 is disposed above thethyroid gland 256 in FIG. 10. In FIG. 10, the battery device 246 isoperatively coupled to a controller with software for generating thepulsed electrical current passing through the body of the patientbetween the anode 248 and the cathode 252. Turning to FIG. 11, it can beseen that the pulsed electrical current (as diagrammatically indicatedby the current lines in FIG. 11) generated by the battery device 246creates an ultrasonic wave 274 from the piezoelectric nanoparticles thatcan be recorded by an ultrasonic transducer 268 located on the skin. InFIG. 11, the ultrasonic transducer 268 is connected to a processor andmonitor 270, which allows an image 272 of the thyroid tumor 266 in FIG.11 to be reconstructed from the ultrasonic wave 274 received at thetransducer 268. In addition, turning to FIG. 12, it can be seen that athyroid tumor 275 may be treated non-invasively with a focusedultrasound beam 284 generated by an ultrasound array transducer 282. Thefocused ultrasound beam 284 passes through the skin and simultaneouslyheats up the tumor tissue and attached piezoelectric nanoparticles to atemperature of 39-40 degrees C. to damage the tumor cells of the tumor275 with the thermal energy and to depolarize the tumor cell membranesby converting the sound waves to an electric pulse to depolarize thetumor cells exposed to the ultrasound, making the cells permeable to themedication/gene used to treat the tumor 275. In FIG. 12, it can be seenthat the heating of the tumor 275 by the focused ultrasound beam 284creates harmonic backscatter ultrasonic waves 286 from the piezoelectricnanoparticles that can be recorded by an ultrasonic transducer 268located on the skin. In FIG. 12, similar to FIG. 11, the ultrasonictransducer 268 is connected to a processor and monitor 270, which allowsan image 276 of the thyroid tumor 275 to be reconstructed from theharmonic backscatter ultrasonic waves 286 received at the transducer268. In FIG. 12, the processor and monitor 270 connected to thetransducer 268 are operatively coupled to another processor 278 thatexecutes software for controlling the energy output of the focusedultrasound delivered by the ultrasound array transducer 282. That is,the processor 278 is operatively coupled to the ultrasound power source280 so as to enable the energy output of the focused ultrasounddelivered by ultrasound array transducer 282 to be selectively variedbased upon feedback from the harmonic backscatter ultrasonic waves 286received by the ultrasonic transducer 268. In this manner, in the systemof FIG. 12, the temperature at the tumor site is able to be selectivelycontrolled by varying the energy output of the focused ultrasounddelivered by ultrasound array transducer 282. Also, in FIG. 12, when thefocused ultrasound is applied to the tumor 275 with piezoelectricnanoparticles attached to the tumor 275, the ultrasound activates thepiezoelectric nanoparticles to produce electrons to depolarize the tumorcells, and the focused ultrasound also creates second harmonic soundwaves 286 that are recorded by the transducer 268 indicating thetemperature at the tumor site. The imaging system 270 in FIG. 12 isconnected to the processor 278 which, in turn, is connected to thefocused ultrasound power source 280 so as to control the temperature ofthe tumor site at 41-43 degrees C. or more, as needed. In this manner,the tumor cells are depolarized and heated simultaneously (i.e.,combining cell thermotherapy with cell depolarization) and medicationconjugated with the piezoelectric nanoparticles is released at the tumorsite so as to damage the tumor cells. Because the tumor cells havealready been rendered generally defenseless by virtue of theirdepolarization, the medication is able to easily pass through thedamaged tumor cell membranes, thus entering the cytoplasms of thedamaged tumor cells and destroying the tumor cells.

In one embodiment, thermotherapy with focused low power focusedultrasound or an alternating magnetic field, etc. is combined with theantibody-coated pluralities of gold, piezoelectric, iron oxide, magneticnanoparticles, nanocage, nanotube, nanoshell, magnetic, paramagnetic,pyroelectric and/or piezoelectric nanoparticles, etc. are coated withthermosensitive polymers, such as chitosan, with quenched fluorescein,liposomes, or polymer micelles or nanoparticles usually poly(N-isopropylacrylamide) carrying a dye another dye or indicator/medication, etc.conjugated with an antineoplastic medication, gene, CRISPR-cas9conjugated with cell penetrating agents or activatable cell penetratingagents injected in the patient's circulation, lymphatic vessels, insidea body cavity, cerebrospinal fluid, etc. The functionalizednanoparticles are exposed to an alternating magnetic field and/orelectrical current generated by a battery where low electrical currentfrom a battery passes from one side of the skin (i.e., the anode)through the body and a lesion or tumor to the cathode electrodepositioned on the opposite side of the skin on the body to raise thetemperature of the piezoelectric or pyroelectric nanoparticles that areinjected inside the body to be attached to the surface antigens of thenormal cells or of tumor cells and heat the nanoparticle/tumor cellcomplexes to 41-43 degrees C., and when exposed to pulses of electricalcurrent with an adjustable signal frequency and voltage, an acousticresponse is produced by electrical stimulation of piezoelectricnanoparticles inside the body that we call electroacoustic sounds orsignals which can be captured with a transducer, or microphone, andconverted to an electrical signal and forwarded to a processor to beconverted to a 1D, 2D, or 3D as an electroacoustic computed tomogramwhile the electrical pulse generated in the piezoelectric nanoparticlesdrives the medication, gene, in the tumor cells locally by depolarizingtheir cell membrane potential and to damage the tumor cells by multiplemodes of the therapy applied non-invasively under observation.

In one embodiment, thermotherapy with focused low power ultrasound oralternating magnetic field, etc. is combined with the antibody coatedpluralities of gold, piezoelectric, iron oxide, nanocage, nanotube,nanoshell, magnetic, paramagnetic, pyroelectric and/or piezoelectricnanoparticles, etc. are coated thermosensitive polymers, such aschitosan, liposomes, or polymer micelles or nanoparticles usuallypoly(N-isopropyl acrylamide) carrying a dye with quenched fluorescein oranother dye or indicator/medication etc. conjugated with anantineoplastic medication, gene, CRISPR-cas9 conjugated with cellpenetrating agents or activatable cell penetrating agents injected inthe patient's circulation, lymphatic vessels, inside a body cavity,cerebrospinal fluid, etc. The functionalized nanoparticles are exposedto a non-focused ultrasound and electrical current generated by abattery where low electrical current from a battery passes from one sideof the skin (i.e., the anode) through the body and a lesion or tumor tothe cathode electrode positioned on the opposite side of the skin on thebody to raise the temperature of the piezoelectric or pyroelectricnanoparticles that are injected inside the body to be attached to thesurface antigens of the normal cells or of tumor cells and createnanoparticle/tumor cells complexes to 41-43 degrees, and when exposed topulses of electrical current with an adjustable signal frequency andvoltage, an acoustic response is produced by electrical stimulation ofthe piezoelectric nanoparticle inside the body that is calledelectroacoustic sounds or signals which can be captured with atransducer, or microphone, and converted to an electrical signal andforwarded to a processor to be converted to a 1D, 2D, or 3D image as anelectroacoustic computed tomogram while the electrical pulse generatedin the piezoelectric nanoparticles drives the medication, gene, in thetumor cells locally by depolarizing their cell membrane potential and todamage the tumor cells by multiple modes of the therapy appliednon-invasively under observation.

In one embodiment, thermotherapy with focused low power ultrasound oralternating magnetic field, etc. is combined with the antibody coatedpluralities of gold, piezoelectric, iron oxide, nanocage, nanotube,nanoshell, magnetic, paramagnetic, pyroelectric and/or piezoelectricnanoparticles, etc. are coated with thermosensitive polymers, such aschitosan, with quenched fluorescein or another dye orindicator/medication etc. conjugated with an antineoplastic medication,gene, CRISPR-cas9 conjugated with cell penetrating agents or activatablecell penetrating agents injected in the patient's circulation, lymphaticvessels, inside a body cavity, cerebrospinal fluid, etc. Thefunctionalized nanoparticles are exposed to a focused low powerultrasound in a non-thermal mode (LIFU) or high power thermal mode(HIFU) generating an electrical current by the focused ultrasound fromthe piezoelectric nanoparticles and/or pyroelectric functionalizednanoparticles, thus creating an electrical signal from the piezoelectricnanoparticles that paralyses the cell permitting the functionalizednanoparticles coated with cell penetrating agents to enter the cellcytoplasm in the cells and release the medication, gene, CRISPR-cas9inside the lesion or tumor by the focused ultrasound to raise thetemperature of the piezoelectric or pyroelectric nanoparticles that areinjected inside the body to be attached to the surface antigens of thenormal cells or of tumor cells and create nanoparticle/tumor cellcomplexes to 41-43 degrees C. and a 1D, 2D, or 3D image as an acousticcomputed tomogram while the nanoparticles drive the medication, gene, inthe tumor cells locally by depolarizing their cell membrane potentialand to damage the tumor cells by multiple modes of the therapy appliednon-invasively under observation.

In one embodiment, where the thermal energy is an electromagneticradiation, the pluralities of nanoparticles absorb the energy andexpand, thereby creating a photoacoustic ultrasound that can be recordedby a photoacoustic or thermoacoustic imaging system indicating thedegree of the temperature achieved, and the increased tumor biomarker inthe circulation after the thermotherapy having an important diagnosticvalue (i.e., presence of a tumor by increased biomarkers) andtherapeutic value for vaccine production after harvesting thecirculating biomarkers and vaccine production with antibody coatednanoparticle with viral-like particles (VLP) or oncolytic viruses, suchas T-Vec, monoclonal antibodies, IL-2, bee toxins or other immunestimulators along with checkpoint inhibitors and administering them withcheckpoint inhibitors, such as PD-1, PD-11, CTLA-4, Jagged 1 inhibitor15D11, etc. and Rock inhibitors, such as Fasudil, or Wnt inhibitors,such as niclosamide, for the future management of the tumor recurrencesin the patient to reactivate the immune response and kill the tumorcells while inducing an immune response by the release of VLP, etc. asstimulators and checkpoint inhibitors enhance the immune response to thetumor locally and also their invisible metastatic lesions elsewhere.

In one embodiment, with reference to FIG. 8, the thermal output of thethermal energy delivery system 210 is a focused high power ultrasound(HIFU) which heats up the tissue/tumor 212 located deep inside the body214 at the focal point of ultrasound using an antibody/medication coatedagent, such as iron oxide gold nanoparticles (GNPs) with activatablecell-penetrating peptides (ACPPs), gold graphene oxides (GOs), gold,silicone, carbon, mixture of magnetic nanoparticles (NPs), gold nanorods(GNRs), gold nanoshells (GNS) or a nanocage filled with a fluoresceindye or located inside a liposome containing quenched fluorescein oranother dye or indicator, etc. that releases the dye when thenanoparticles temperature reaches a temperature of about 41-43 degreesC., thereby releasing the fluorescein in the circulation that can beobserved by taking a blood sample or radiating the nail bed 218 of thepatient with UV radiation delivered via a fiber optic 220 so that nownon-quenched fluorescein fluoresces as green light passing through ablue filter 222 (see FIG. 8) demonstrating a hand held system with ablue wave length radiating source, such as a laser or diode 228providing a wavelength of 380-390 nm illuminating a finger nail 218 andthe capillaries in the nail bed, which in presence of unquenchedfluorescein or another dye or indicator, etc. fluoresces, etc. a greenlight of 410 to 420 nm passing through a blue absorbing filter 222 andreached through a fiber optic 224 and a photomultiplier that convertsthe light to an electrical signal, then to a processor 226 that, inturn, is connected to the thermal delivery system indicating thetemperature of 41-43 degrees C. is achieved, the operator can viasoftware maintain the energy delivery for the desired time (e.g., 1-10minutes or more, if needed) or continue the energy delivery until thenext level of 43-56 degrees C. when a focused ultrasound is used and itsterminal is connected to the same processor to continue deliveringenergy to reach the temperature of 56 degrees C. creating a popping orcavitation sound at the boiling point of the PFCL Nps when theultrasonic receiver or the microphone 216 records a cavitation sound tobe reported, via a receiver, to the processor 226 connected to eitherthe ultrasonic unit or the AMF unit to stop producing the thermal energyto the tissue.

In one embodiment, a patient with a malignant tumor or benign lesion isadministered antibody coated magnetic nanoparticles conjugated with Rockinhibitors, or Wnt inhibitors, or GSK 269962 inhibitors conjugated witha thermosensitive polymer, such as PEG, or chitosan or other polymers ornanoparticles carrying the slow release medication from porous silicon,or polylactic acid, or polyglycolic acid, along with other medicationsor genes with CRISPR-cas9 that may be released under an alternatingmagnetic field (AMF) at a 100 kHz to 300 kHz to shake up the polymericmaterial, gene, and medication without significant thermal effect orincreasing the alternating magnetic field to 1 MHz to 10 MHz or more toinduce simultaneously a thermal energy to heat up the tissue to adesired temperature as a result of the AMF frequency. In one embodiment,the thermal energy increases the surrounding tumor temperature dependingon the current of AMF, the concentration of antibody/aptamer conjugatedMNP and the time of application to achieve a temperature from 37-41degrees C. or 41-50 degrees C. or more which can be calculated by asoftware depending on the AMF frequency and the duration of the therapyto release the gene or medication and damage the tumor cell withincreasing temperature or killing them.

In one embodiment, the external thermal energy is delivered by acombination source, such as electromagnetic radiation, alternatingmagnetic field, radio frequency (RF), and low power focused ultrasoundeither in a compressive or pressure or high power thermal mode and thenanoparticle/tumor temperature can be imaged simultaneously with theultrasound, MRI, CT-scan, PET-scan, photoacoustic imaging unit, etc. orthe change in CBE of acoustic harmonics (hCBE), generated by nonlinearpropagation of the ultrasound beam in the tissue or a combinationthereof to image the temperature.

In one embodiment, monoclonal antibodies (mAb) coated nanoparticlesadministered intravenously with checkpoint inhibitors, andantineoplastic medication, taxol derivatives or Wnt inhibitors, such asWIKI4, are conjugated with thermosensitive polymers coated pluralitiesof nanoparticles with activatable cell-penetrating peptides (ACPPs),nanoshells or nanocages, etc. to deliver monoclonal antibodies locallyto the tumor cells while heating with a source of energy to melt thethermosensitive coating of the nanoparticle at the temperature of 41-43degrees C. releasing the medications, and reducing the systemic sideeffects of intravenously or subcutaneously, intra-arterially, locally,in the body cavities, bladder, nose mouth, cerebrospinal fluidintraperitoneally delivered mAbs, checkpoint inhibitors and immunestimulators, Rock inhibitors, such as Fasudil, etc., mAbs, or utomilumabantineoplastic medication, and removing them along with the releasedtoxins (i.e., the toxic cytokines released from the tumors out ofcirculation) using dielectrophoresis, plasma exchange, plasmapheresis,blood dialysis at the end of the therapy, etc. and systemic medicationwith tocilizumab, a monoclonal antibody that targets interleukin-6 andornithine phenylacetate an ammonia scavenger, or low molecular weightheparin for treatment of hepatic encephalopathy, a neuropsychiatricsyndrome associated with hyperammonemia or ARF6 inhibition.

In one embodiment, the method of providing antibody -coatednanoparticles/gemtuzumab to a site, or for systemic delivery, may becombined with immunologically based B-cells or T-cells that areoptionally genetically modified. Such B-cells and T-cells may attacklocalized and/or unlocalized cancer cells or hematological cancers(e.g., leukemias, lymphomas, etc.). In one embodiment, extra-corporaldialysis and/or plasmapheresis and systemic medication with tocilizumab,a monoclonal antibody that targets interleukin-6 and ornithinephenylacetate an ammonia scavenger, for treatment of hepaticencephalopathy, a neuropsychiatric syndrome associated withhyperammonemia in combination with the inventive method of deliveringantibody-coated nanoparticles with ACPP to accomplish removal ofexcessive tumor protein in order to protect vital organs (e.g., kidney,liver, brain). In one embodiment, one or more of these therapies areemployed at lower doses when used in combination with the method ofdelivering antibody/medication coated nanoparticles.

In one embodiment, the application of thermotherapy to reach thetemperature of 41-43 to 50 C. to 56 degrees C. temperature releases morecytokines from the damaged tumor cells activating the immune cellresponse more than if the medications were administered alone withoutthermotherapy, eliminating the need for CAR-T cellular therapy whichrequires a time consuming and expensive preparation with the chance ofincreased toxicity and an autoimmune response.

In one embodiment, monoclonal antibodies, checkpoint inhibitors orantineoplastic medications, utomilumab are conjugated with antibodycoated nanoparticles, nanoshells nanocage magnetic or paramagnetic, andVLP or oncolytic viruses, such as T-Vec, monoclonal antibodies, IL-2,bee toxins or other immune stimulators along with checkpoint inhibitorssuch as PD-1, PD-L1, CTLA-4, Jagged 1 inhibitor 15D11, etc. to deliverthem to the tumor cells by systemic or local administration, releasingthem from the thermosensitive polymers of the pluralities ofnanoparticles with activatable cell-penetrating peptides (ACPPs) at acontrolled predetermined temperature of 41-43 degrees C. sinceintravenous administration of monoclonal antibodies or check pointinhibitors or antineoplastic medications or immune stimulators areadministered in significantly higher dose than nanoparticle conjugatedmedications, VLP, and immune stimulator that are released locally at thecancer cells and have many side effects such as hyperactivation ofT-cells and cytokine release causing nonspecific or specific bodyresponse (e.g., checkpoint inhibitors and vaccine therapy causerecruitment of specific T-cell to attack the tumor and potentially somespecific organs that share the same checkpoint inhibitors) or causeexcessive cytokine release producing Cytokine release syndrome (CRS)associated with fever, tachycardia, vascular leak, oliguria,hypotension, hyper activation of T-cells and cytokine such as those seenwith checkpoint Protein Inhibitors, Rock inhibitors, such as Fasudil,and its derivatives etc., or Wnt inhibitors Ipilimumab blocks CTLA-4;pembrolizumab blocks PD-1; and nivolumab also blocks PD-1, or utomilumabinducing colitis, pancreatitis, pneumonitis, hepatitis, skin reactionsor mAbs induce arthralgia, enteritis, encephalitis, Guillain-Barresyndrome, myasthenia gravis-like syndrome, and autoimmune bone marrowsuppression, skin-related toxicities, hepatitis and endocrinopathies andoften multiple therapy, when cytokines are used, such as Recombinanthuman interferon alfa (IFN), one observes fever, fatigue, headache andmyalgia, thrombocytopenia and leukopenia, Vitiligo, lupus, rheumatoidarthritis, polymyalgia rheumatica, autoimmunity, neurotoxicity, andmyocarditis and toxicities related to adaptive cell therapy and toenhance immune therapy.

In one embodiment, a combination of nanoparticles with activatablecell-penetrating peptides (ACPPs) is used to have additionalfunctionalities as well as imaging, thermoacoustic imaging forthermotherapy and imaging and tumor therapy by administering to apatient systemically or locally either antibody/medication coated coppersulfide (CuS) nanoparticles, or polymeric microbubbles of Polylacticacid or lactic acid microcapsules employing the water-in-oil-in-waterdouble emulsion method or gold Nanorod-loaded PLA microcapsules forcombined ultrasound contrast imaging and conjugated with athermosensitive polymer, such as chitosan with DNA, siRNA, RNAi, withCRISPR enzyme or with or without macrolides, such as cyclosporine A,mycophenolic acid, tactolimus or ascomycin or other drugs delivery at41-43 degrees C. temperature or monoclonal antibodies check pointinhibitor, anti-neoplastic medications, or antibody coated goldnanoparticles with activatable cell-penetrating peptides (ACPPs) orantibody/medication coated gold nanoshells Perfluoro carbon liquid, orantibody coated perfluorohexane (PFH)-loaded magnetic hollow iron oxidenanoparticles (HIONs) and Fe—O stretching vibration mode of Fe3O4T, orantibody coated nanoparticles, such as antibody/medication coated SPIOsor Gold Nanorod and iron oxide coating nanocapsules that absorb thermaldelivery using alternating magnetic field, focused high power ultrasoundand electromagnetic radiation or imaging simultaneously with MRI or CTcontrast imaging, PET-scan, while having high X-ray absorptioncoefficient, to have multifunctional capabilities for cancer diagnosisand thermotherapy and thermoacoustic imaging for control of thetemperature.

In one embodiment, for administration inside the tumor or in thecirculation, the thermosensitive polymeric/medication compound ischitosan or liposomes or liposome-coated chitosan or polycaprolactone,polyanhydrides or polyesters conjugated with monoclonal antibodies(mAbs) or checkpoint inhibitors, Rock inhibitors such as Fasudil, etc.,VLP, or oncolytic viruses, monoclonal antibodies, IL-2, bee toxins orother immune stimulators along with checkpoint inhibitors, such as PD-1,PD-L1, CTLA-4, Jagged 1 inhibitor 15D11 or an antineoplastic medication,daunorubicin, ara-C or cytarabine, taxol, taxane derivatives,doxorubicin etc. that are released along with a quenched fluorescein dyeor another quenched dye or indicator/medication, PFC nanobubbles, afterthermal energy application using laser, focused ultrasound, AMF,microwaves, or radiofrequency radiation at the temperature of 41-43degrees C., etc., the medication or dye is released in the circulationand can be seen in a few seconds in the circulation using a blood sampleor inside a transparent butterfly tubing that fluoresces under UVradiation indicating presence of the dye indicating that the temperature41-43 degrees C. at the site of the heated nanoparticles/tumor isachieved and the increased tumor biomarkers in the circulation after thethermotherapy has an important diagnostic value (i.e., confirming thepresence of a tumor) and therapeutic value as biomarkers for the futuremanagement of the patient with recurrences by making a vaccine using thebiomarkers and VLP, IL-2 conjugated with antibody coated nanoparticlesand administering them with checkpoint inhibitors, such as PD-1, PD-L1,CTLA-4, Jagged 1 inhibitor 15D11, etc. to enhance immune therapy, andRock inhibitors, such as Fasudil or Wnt inhibitors, such as niclosamide,to attach to the potential tumor cells, while applying thermal energy todamage recurrence of the tumor and induce reactivation of the immuneresponse to eliminate them, thereby eliminating the need to repeat CAR-Tcell therapy unless the patient is immunosuppressed.

In one embodiment, the administered thermosensitive polymeric compoundis chitosan conjugated with mAbs or checkpoint inhibitors, orantineoplastic medication, Daunorubicin, ara-C or cytarabine, taxol,taxane derivatives, etc. that are released at the temperature of 41-43degrees C. along with a quenched fluorescein dye or another quenched dyeor indicator, etc. in the circulation and can be seen in a few secondsin the circulation using a blood sample or inside a transparentbutterfly tubing that fluoresces under UV radiation indicating presenceof the dye indicating that the temperature 41-43 degrees C. at the siteof the heated nanoparticles/tumor is achieved and the increased tumorbiomarkers in the circulation after the thermotherapy has an importantdiagnostic value (i.e., confirming the presence of a tumor) andtherapeutic value as biomarkers for the future management of the patientwith recurrences by making a vaccine using the biomarkers and VLP oroncolytic viruses, monoclonal antibodies, IL-2, bee toxins or otherimmune stimulators along with checkpoint inhibitors, such as PD-1,PD-L1, CTLA-4, Jagged 1 inhibitor 15D11 conjugated withantibody/medication coated nanoparticles and administering them withRock inhibitors, such as Fasudil, or Wnt inhibitors, such asniclosamide, to attach to the potential tumor cells, while applyingthermal energy to prevent recurrence of the tumor and inducereactivation of the immune response to eliminate them as enhanced immunetherapy, thereby eliminating the need to repeat CAR-T cell therapyunless the patient is immunosuppressed.

In one embodiment, the administered thermosensitive polymeric compoundis chitosan conjugated with mAbs or check point inhibitors, orantineoplastic medication, Artemisinins or artemisinin analogues againsta variety of cancer cells, promoting apoptosis, preventing angiogenesisinhibition of Toll-like receptors, Syk, tyrosine kinase inhibitor,phospholipase Cγ, PI3K/Akt, MAPK, STAT-1/3/5, NF-κB, Sp1, Axitinib andNrf2/ARE signaling pathways that are released at the temperature of41-43 degrees C. using photoacoustic or after thermal energyapplication, such as laser, focused ultrasound, AMF or microwave orradiofrequency radiation at the temperature of 41-43 degrees C. alongwith a quenched fluorescein dye or another quenched dye or indicator,etc. in the circulation and can be seen in a few seconds in thecirculation using a blood sample or inside a transparent butterflytubing that fluoresces under UV radiation indicating presence of the dyeand imaging the temperature by the change in backscattered energy of thesignal (CBE), the backscattered radio-frequency (RF) echo-shift due tochange in the speed of sound and thermal expansion of the medium,indicating that the temperature 41-43 degrees C. has been achieved atthe site of the heated nanoparticles/tumor and the increased tumorbiomarkers in the circulation after the thermotherapy has an importantdiagnostic value (i.e., confirming the presence of a tumor) andtherapeutic value as biomarkers for the future management of the patientwith recurrences by making a vaccine using the biomarkers and VLP, oroncolytic viruses such as T-Vec, monoclonal antibodies, IL-2, bee toxinsor other immune stimulators conjugated with antibody coatednanoparticles and administering them with checkpoint inhibitors, such asPD-1, PD-L1, CTLA-4, Jagged 1 inhibitor 15D11, to enhance immunetherapy, etc., and Rock inhibitors, such as Fasudil, or Wnt inhibitors,such as niclosamide, to attach to the potential tumor cells whileapplying thermal energy to damage recurrence of the tumor and inducereactivation of the immune response to eliminate them, therebyeliminating the need to repeat genetically modified CAR-T cell therapyunless the patient is immunosuppressed while antibody coatednanoparticles/checkpoint inhibitors and VLP attach to the localized orcirculating tumor cells and their exosomes, which are carrying acheckpoint protein, such as PD-L1 (to disguise themselves), and thetumor cells are recognized by the T cells, which together with killercells phagocytose them, and enhance the immune response to the tumor andits exosomes and the circulating cells.

In one embodiment, the Wnt/β-catenin pathway can be inhibited indirectlyby inhibiting GSK-3 Glycogen synthetizing Kinase, such as lithiumchloride, a simple salt, or its Phosphoaminophosphonic form at anextremely low concentration of 1-3 nano-micromolar that can only bedelivered using antibody coated pluralities of nanoparticles conjugatedwith thermosensitive polymers, such as chitosan conjugated with ACPP andquenched fluorescein, or another quenched dye or indicator/medication,etc. and or an antineoplastic medication or monoclonal antibody torelease the dye and medication by thermal energy at controlledtemperature of 39-41 degrees C. to not only inhibit the Wnt pathway butalso treat various Wnt/β-catenin signaling in a number of diseases suchas inflammation, brain cancers, glioblastoma, glioma, prostate, breast,colon cancer, ovarian cancer, and Alzheimer's disease.

In one embodiment, the Wnt/β-catenin pathway inhibitors can be used withsulfasalazine inhibitor of PIK3CA mutations, enzymes that are involvedin cellular growth control signals, that is known to inhibit xCT, andcan be delivered using antibody coated pluralities of nanoparticlesconjugated with thermosensitive polymers, such as chitosan, conjugatedwith ACPP and quenched fluorescein, or another quenched dye orindicator/medication, etc. and or an antineoplastic medication ormonoclonal antibody to release the dye and medication by thermal energyat a controlled temperature of 41-42 degrees C. to not only inhibit theWnt pathway but also treat various Wnt/β-catenin signaling in a numberof diseases inhibit cCT and PIK3CA, such as in inflammation, braincancers, glioblastoma, glioma, prostate, breast, colon cancer, andovarian cancer.

In one embodiment, the Wnt/β-catenin pathway inhibitors or Rockinhibitors, such as Fasudil, etc., can be used with sulfasalazineinhibitor of PIK3CA mutations, enzymes that are involved in cellulargrowth control signals, that is known to inhibit xCT and can bedelivered using antibody coated pluralities of nanoparticles conjugatedwith thermosensitive polymers, such as chitosan, conjugated with ACPPand quenched fluorescein or another quenched dye orindicator/medication, such as inhibition of tyrosine, monoclonalantibodies to EGFR, VEGFR, PDGFR, and c-kit Cdc42: cell division controlprotein 42, ERK: extracellular signal-regulated kinase, mTOR: mammaliantarget of rapamycin, PI3K: phosphatidylinositol 3-kinase, EGF(R):epidermal growth factor (receptor), Grb2: growth factor receptor-boundprotein 2, JNK: c-Jun N-terminal kinase, MEK/MKK: mitogen-activatedprotein kinase kinases, PDGF(R): platelet derived growth factor(receptor), SOS: son of sevenless, TAK: TGFβ-activated kinase, TGF:transforming growth factor, and VEGF(R): vascular endothelial growthfactor (receptor) to release the dye and medication by thermal energy atcontrolled temperature of 41-42 degrees C. to not only inhibit the Wntpathway but also treat various Wnt/β-catenin signaling in a number ofdiseases inhibit cCT and PIK3CA such as in inflammation, brain cancers,glioblastoma, glioma, glioma multiforme, prostate, breast, colon cancer,ovarian cancer.

In one embodiment, the Wnt/β-catenin pathway inhibitors, such asniclosamide at 500 mg dose, ivermectin 1 gram-2 gram once a week, etc.,or Rock inhibitors, such as Fasudil at 40-80 mg, etc., can be usedorally at approved doses or systemically with systemic sulfasalazineinhibitor of PIK3CA mutations, enzymes that are involved in cellulargrowth control signals, that is known to inhibit xCT and can bedelivered using antibody coated pluralities of nanoparticles conjugatedwith thermosensitive polymers, such as chitosan, conjugated with ACPPand quenched fluorescein or another quenched dye orindicator/medication, such as inhibition of tyrosine, monoclonalantibodies to EGFR, VEGFR, PDGFR, and c-kit Cdc42: cell division controlprotein 42, ERK: extracellular signal-regulated kinase, mTOR: mammaliantarget of rapamycin, PI3K: phosphatidylinositol 3-kinase, EGF(R):epidermal growth factor (receptor), Grb2: growth factor receptor-boundprotein 2, JNK: c-Jun N-terminal kinase, MEK/MKK: mitogen-activatedprotein kinase kinases, PDGF(R): platelet derived growth factor(receptor), SOS: son of sevenless, TAK: TGFβ-activated kinase, TGF:transforming growth factor, and VEGF(R): vascular endothelial growthfactor (receptor) to release the dye and medication by thermal energy atcontrolled temperature of 41-42 degrees C. to not only inhibit the Wntpathway but also treat various Wnt/β-catenin signaling in a number ofdiseases inhibit cCT and PIK3CA such as in inflammation, brain cancers,glioblastoma, glioma, multiforme, prostate, breast, colon cancer, andovarian cancer, etc.

In one embodiment, one achieves a theranostic or diagnostic andtherapeutic effect when the antibody coated pluralities ofantibody/aptamer-conjugated nanoparticles with activatablecell-penetrating peptides (ACPPs) conjugated with a Wnt inhibitor, suchas ivermectin, and a Rock inhibitor, such as Fasudil, Y-27632, smallmolecule inhibitor of ROCK1 and ROCK2 which act as an anti-inflammatoryagent and inhibit Wnt activation, are encapsulated in the antibodycoated liposomes or antibody coated nanoparticles conjugated with athermosensitive polymer such as chitosan, and containing an immunestimulators, such as VLP, oncolytic viruses, IL-2, Interferons, TLR 7,TLR8, checkpoint inhibitor(s), polysaccharide K, a toxin and aphotosensitizer such as Verteporfin or phthalocyanine, etc. or anotherdye and electromagnetic energy, such as laser, absorbing antibody coatednanoparticles with activatable cell-penetrating peptides (ACPPs), suchiron oxide, gold, silicon carbon, magnetic, non-magnetic NPs ornanoshells, nanocages, nanotubes, nanorods, nanospheres, or any othershells produced (e.g., polylactic or polyglycolic acid, etc.) orpiezoelectric nanoparticle administered systemically, topically, locallyinside the tumor exposed to a thermal energy source such ultrasound oras laser light from 360-1000 nm or more or microwave or Radio frequencywave, to increase the temperature of the nanoparticles inducing aphotoacoustic or thermoacoustic sound measuring the temperature of theheated tumor from 40-43- or more as needed and, the lipid component ofthe liposome and chitosan, melts, releasing medication and the dye attemperature of 41-43 C. degrees C. or more to release the medication,etc. initiating a precise photodynamic effect on the tumor cells alongwith VLP and checkpoint inhibitors release inducing a photodynamiceffect with thermotherapy and immune stimulation to kill and remove thetumor cells by the natural killer cells, activated T-cells etc., thetemperature of 41-43 or more degrees C. is achieved and the medicationis released from the NPs, and the achieved temperature is recognized byphotoacoustic acoustic imaging for control of the temperaturecommunicating with the thermal delivery e.g. laser unit via a processorto control the amount of energy released by the thermal energy deliveryunit via a processor to stop or increase or decrease the temperature orby increasing the temperature of the nanoparticles or nanoshells, whileantibody coated nanoparticles/checkpoint inhibitors and VLP attach tothe localized or circulating tumor cells and their exosomes, which arecarrying a checkpoint protein, such as PD-L1 etc. (to disguisethemselves), and the tumor cells and exosomes are recognized by theT-cells, which together with natural killer cells phagocytose them, andenhance immune response to the tumor, and its exosomes and thecirculating cells.

In one embodiment, these biguanides administered along with aspirin canprevent cancer formation and in cancer treatment by inhibition of mTORsignaling via AMP-activated protein kinase (AMPK)-dependent and-independent path and inhibition of COX-1/COX-2 and modulation of theNFκB or STAT3 pathway and activation AMPK, and both agents may affectNotch, Wnt/β-catenin when delivered through the thermosensitivepolymeric coated functionalized pluralities of nanoparticles and ACPPintravenously, orally or intracavity or intra-arterially at one nanogramto 100 nanogram concentrations interfering with the glucose metabolismof the tumor stem cells proliferation causing them to starve.

In one embodiment, antibody/medication coated pluralities ofnanoparticles administered with activatable cell-penetrating peptides(ACPPs) conjugated with Wnt inhibitor such as FH535, or IWP-2, orPNU-74654, or IWR-1endo, or IWR-exo, or demethoxycurcumin, or CCT036477,or KY02111, or ivermectin, niclosamide, metformin, or phenformin areencapsulated in nanogram concentrations within the chitosan coating ofthe nanoparticles, such iron oxide, gold, silicon carbon, magnetic,non-magnetic Nps or nanoshells, nanocages, nanotubes, nanorods,nanospheres, etc. inside the liposomes containing fluorescein andelectromagnetic energy absorbing nanoparticles. When exposed to athermal energy source such as, electromagnetic radiation, microwave,radiofrequency radiation, focused high power ultrasound, or low powerfocused ultrasound (LIFU) or AMF and antibody/aptamer-conjugated MNPwith liposomes where the lipid component of the liposome melts attemperature of 40-43 degrees C. releasing the medication locally or inthe circulation and simultaneous imaging the temperature at 41-43degrees C. to heat up the tumor and the increased tumor biomarkers inthe circulation after the thermotherapy has an important diagnosticvalue (i.e., confirming the presence of a tumor) and therapeutic valueas biomarkers for the future management of the patient with recurrencesby making a vaccine using the biomarkers and VLP, IL-2 conjugated withantibody coated nanoparticles and checkpoint inhibitors, such as PD-1,Jagged 1 inhibitor 15D11, to attach to the potential tumor cells whileapplying thermal energy to damage recurrence of the tumor and inducereactivation of the immune response to eliminate them, while antibodycoated nanoparticles/checkpoint inhibitors and VLP attach to thelocalized or circulating tumor cells and their exosomes, which arecarrying a checkpoint protein, such as PD-L1 (to disguise themselves),and the tumor cells are recognized by the T cells, which together withkiller cells phagocytose them, and enhance the immune response to thetumor and its exosomes and the circulating cells and eliminate them.

In one embodiment, thermotherapy with focused ultrasound or alternatingmagnetic field, etc. is combined with antibody coated pluralities ofmagnetic nanoparticles (MNP) are administered with activatablecell-penetrating peptides (ACPPs) conjugated with a Wnt inhibitor, suchas FH535, or IWP-2, or PNU-74654, or IWR-1endo, or IWR-exo, ordemethoxycurcumin, or CCT036477, or KY02111, or ivermectin, niclosamide,metformin, or phenformin with and without syrosingopine are encapsulatedin nanogram concentrations within the chitosan coating of thenanoparticles, such as iron oxide, gold, silicon, carbon, magnetic,non-magnetic nanoparticles or nanoshells, nanocages, nanotubes,nanorods, nanospheres, etc. inside the liposomes containing medicationetc. and electromagnetic energy absorbing nanoparticles. When exposed toa thermal energy source, such as electromagnetic radiation, microwaves,radiofrequency radiation, high power focused ultrasound, compressive orpressure low power ultrasound or AMF, the lipid component of theliposome melts at a temperature of 40-41 degrees C., thus releasing themedication etc. locally or in the circulation.

In one embodiment, the photoacoustic or thermoacoustic unit iscommunicating with the thermal delivery unit via a processor to controlthe amount of energy released by the thermal energy delivery unit via aprocessor to stop or increase or decrease the temperature all below thethermal denaturation temperature of proteins which is 60 degrees C. atthe site of the nanoparticles as desired to prevent excessive thermaldamage to the normal surrounding cells.

In one embodiment, when the parameters of thermal heating are keptconstant to prevent excess heating of the nanoparticles with activatablecell-penetrating peptides (ACPPs)/tumor is reached at 41-43 degrees C.when the quenched fluorescein is used with a thermosensitive polymer orlipid or liposomes having temperature triggered opening of a leucinezipper peptide inserted in the membrane of a dye and medication carryingliposome opens a channel through which the drug is released at thetemperature or 41-43 degrees C. or opens drug-permeable pores createdbubble formation from the decomposition of encapsulated ammoniumbicarbonate and the fluorescent dye is recognized by an imaging unitcommunicating with the thermal delivery unit via a processor to controlthe amount of energy released by the thermal energy delivery unit via aprocessor to stop or increase or decrease the temperature or thetemperature reaches about 56 degrees C. when the perfluorohexane (PFH)or perfluoropentane incorporation in the nanocage or nanoshellevaporates, thus causing an acoustic sound that can be recorded with anultrasonic receiver that is below 60 degrees C. of protein denaturation.

In another embodiment, the thermosensitive polymers are lipids,liposomes, micelles, polycaprolactone, polyanhydrides, chitosan,poly-lactic acid (PLA), poly(ethylene glycol), ethyl ethermethacrylate-copoly(ethylene glycol), methyl ether methacrylate,Pluronic F127 (F127)[, poly(methylmethacrylate) (PMMA)],poly-n-isopropylacrylamide (PNIPAM), crosslinked ONIPAN hydrogels loadedwith Fe3O4 nanoparticles, poly(ethyleneimine)-modified poly(ethyleneoxide)-poly(propylene oxide)-poly(ethylene oxide) (PEO-PPO-PEO) blockcopolymer and nanoparticle-assembled capsules (NACs) usingPoly(allylamine hydrochloride), andchitosan-poly(N-isopropylacrylamide).

In one embodiment, the thermosensitive polymers with, for example, knownpH sensitive polymers, magnetically controlled release agents,magnetoliposome agents, thermosensitive liposomes entrapping iron oxidenanoparticles with activatable cell-penetrating peptides (ACPPs), etc.are used, to achieve controlled drug release with external thermaldelivery such as microwave, radio frequency (RF) or focused ultrasound,etc. under the control of a processor at a temperature of 41-43 degreesC.

In one embodiment, variable architecture thermosensitive polymers thatare temperature- and pH-responsive, smart polymers, including syntheticcopolymers and polymer derivatives, functionalized pluralities ofnanoparticles encased in poly(N-isopropylacrylamide) (PNIPAM),poloxamers, poloxamines, polymers with thiol groups (PMA.sub.SH) areused with antineoplastic medications or monoclonal antibodies to releasethe medication with an external source of energy or magnetic inductionunder the control of a processor at temperature of 41-43 degrees C.

In one embodiment, the pluralities of functionalized pluralities ofnanoparticles with activatable cell-penetrating peptides (ACPPs) andtemozolomide are administered intravenously, intra-arterially, locally,etc. contain at least one radioactive isotope and coated with anti-tumorantibodies, monoclonal antibody, the nanoparticles forming an antibodylabeled nanoparticle-cell complex at a target site, the radioactivenanoparticle resulting in radiation therapy at a dose sufficient todamage tumor-associated vessels at the localized target site, and theDNA of the tumor stem cells while administering thermal energysimultaneously to achieve controlled drug release with external thermaldelivery such as microwave, RF, or low power ultrasound etc. under thecontrol of a processor at temperature of 41-43 degrees C. in treatmentof many brain tumors specially glioblastoma.

In one embodiment, the pluralities of radioactive functionalizednanoparticles with activatable cell-penetrating peptides (ACPPs) coatedwith thermosensitive polymers and antineoplastic medication, such astaxol and or temozolomide, are administered locally or systemically,intravenously, intra-arterially, inside a body cavity, and are magnetic,diamagnetic, ferromagnetic, non-ferromagnetic and/or paramagneticnanoparticle, wherein the radioactive isotope is an alpha.-emittingisotope, a beta.-emitting isotope, or a combination thereof consistingof At.sup.211, Ac.sup.225, Bi.sup.212, Bi.sup.213, Ra.sup.223,Pb.sup.212, Tb.sup.149, I.sup.131, Cu.sup.64, I.sup.131, Bi.sup.213, andBi.sup.212, with the dose of the isotope ranges from 10 .mu.Ci to 20,000.mu.Ci, and thermal energy is administered simultaneously to achievecontrolled drug release with an external or internal thermal deliverysuch as laser, microwave, RF, or low power focused ultrasound, etc.under the control of a processor at a temperature of 41-43 degrees C. todamage the tumor cells and their vascular supplies, the DNA of the tumorstem cells, and release the medication, such as temozolomide intreatment of many cancers including brain tumors.

In one embodiment, the pluralities of functionalized radioactive gold orcombination of gold and ferric oxide nanoparticles with activatablecell-penetrating peptides (ACPPs) coated with thermosensitive polymersand checkpoint inhibitors, such as ipilimumab nivolumab, Rockinhibitors, such as Fasudil, etc., bevacizumab, and/or an antineoplasticmedication, such as taxol and or temozolomide, are administered locallyor systemically, intravenously, intra-arterially inside a body cavity,where magnetic, diamagnetic, ferromagnetic, non-ferromagnetic, and/orthe paramagnetic nanoparticle is an interstitial brachytherapynanoparticles beta radiators and thermal energy is administeredsimultaneously to achieve controlled drug release with an external orinternal thermal delivery such as laser, microwave, RF, or high powerultrasound, etc. under the control of a processor at a temperature of41-43 degrees C. to damage the tumor cells and their vascular supplies,the DNA of the tumor stem cells, and release the medication, such astemozolomide, in treatment of many cancers including brain tumors wherethermal therapy can be repeated in the postoperative period as needed.

In one embodiment, groups of non-magnetic, paramagnetic, or magneticfunctionalized pluralities of nanoparticles with activatablecell-penetrating peptides (ACPPs) and thermosensitive polymeric coatingare produced where polymeric coating of these nanoparticles renders themless toxic to normal tissue such as Iodine.sup.131 may be crosslinkedwith antibody-coated nanoparticles to label the tumor or bacterialcells, both for therapy and imaging. Anti-integrins or anti-VEGFs mayalso be incorporated or the nanoparticles may be used simultaneously fordrug delivery purposes against these tumor or bacterial cells, with thedrug in PDA, PGLA, dextran, dendrimers, PEG, etc.

In one embodiment, a focused high energy ultrasound is used to heat theantibody/medication coated nanoparticle with activatablecell-penetrating peptides (ACPPs) conjugated with thermosensitivepolymeric coating carrying a medication, dye, monoclonal antibody, orcheck point inhibitors alone to release the medication when atemperature of 43 degrees C. has been achieved and the combination ofmonoclonal antibody and thermotherapy eliminate the need for the use ofgenetically modified CAR-T cell that increases postoperative cytokinestorm and autoimmune response, and the increased tumor biomarkers in thecirculation after the therapy has an important diagnostic value (i.e.,indicating presence of a tumor) and therapeutic value as biomarkers forproduction of vaccine and administering them with checkpoint inhibitorssuch as PD-1, PD-11, CTLA-4, Jagged 1 inhibitor 15D11, etc. to enhanceimmune therapy and oral Rock inhibitors, such as Fasudil, (40-80 mgtablets) etc. or Wnt inhibitors, such as niclosamide (<500 mg dose),ivermectin etc. 1-2 gram dose once in a week or can be repeated asneeded in another month for the future management of the tumorrecurrences in the patient, while the Rock inhibitor reduce TGF-βproduction after therapy and the subsequent scar formation.

In one embodiment, the temperature of the heated functionalizedliposomes/nanoparticles/tumor cells are measured when a focused lowpower focused ultrasound and alternating magnetic field and MNP is usedto heat up the nanocage, or nanoshells to up to the boiling point oftheir PFCL hexane or pentane that is about 50 to 56 degrees C. and imagethe temperature with thermoacoustic imaging system for control of thetemperature or produce the change in backscattered energy of the signal(CBE), the backscattered radio-frequency (RF) echo-shift due to changein the speed of sound and thermal expansion of the medium.

In one embodiment, either of the temperature indicators communicate viaa processor that the desired temperature is achieved by a given thermalenergy, to control the temperature and the duration of the desiredtemperature in the tissue, thereby separating the thermal drug deliveryprocess from the cells destructive thermal delivery, as needed, via aprocessor that connects the temperature imaging system to the thermaldelivery unit.

In one embodiment, the antibody/medication coated nanoparticle ornanoshell solution containing 2.67 nM gold nanorods, 2% human serumalbumin, 0.04% (w/v) avidin and filling with PFC (C 3F 8) gas, thenmodified via biotin-avidin technique to result in anti VEGF (Avastin),aflibercept to target by ultrasonography, the area of angiogenesis, suchas a tumor or wet form of age related macular degeneration The GNRscould induce photoacoustic or thermoacoustic imaging and thermal therapyunder electromagnetic radiation or focused low power ultrasound showingthe theranostic value of this modality on keeping the temperature at thedesired temperature of 41-43 degrees C. not to damage the surroundingnormal tissue while treating a tumor (e.g., glioblastoma) or ARMD at lowtemperature and release medication and Wnt inhibitor of Rock inhibitors(Fasudil, Y-27632, small molecule inhibitor of ROCK1 and ROCK2 which actas an anti-inflammatory agent and inhibit Wnt activation, from thethermosensitive nanoparticle under the control of the temperature andthe increased glioblastoma biomarkers, such as co-receptor, glypican 2(GLP2) or cell surface GPC2 in the circulation after the therapy has animportant diagnostic value (i.e., indicating the presence of a tumor)and therapeutic value as biomarkers for vaccine production andadministering them with antibody coated VLP, checkpoint inhibitors, suchas PD-1, PD-11, CTLA-4, Jagged 1 inhibitor 15D11, etc. to enhance immunetherapy and Rock inhibitors, such as Fasudil etc., or Wnt inhibitors,such as niclosamide, for the future management of the patient in case ofthe recurrences.

In one embodiment, the antibody/medication coated nanoparticle ornanoshell solution containing 2.67 nM gold nanorods, 2% human serumalbumin, 0.04% (w/v) avidin and filling with PFC (C 3F 8) gas, thenmodified via biotin-avidin technique to result in anti VEGF (Avastin),aflibercept or Axitinib and quenched fluorescein, bubble liposomescarrying fluorescein/medication which contain air pockets ornanoemulsions of PFC, gen or another dye or indicator, in athermosensitive coating of the functionalized pluralities ofnanoparticles with activatable cell-penetrating peptides (ACPPs) totarget by ultrasonography the area of angiogenesis, such as tumor orsuspicious breast cancer or ovarian cancer, while GNRs could inducephotoacoustic or thermoacoustic imaging and thermal therapy under AMFwith MNP or focused low power ultrasound showing the theranostic valueof this modality on keeping the temperature at the desired temperatureof 41-43 degrees C., imaging the temperature with the change inbackscattered energy of the signal (CBE), the backscatteredradio-frequency (RF) echo-shift due to change in the speed of sound andthermal expansion of the medium, so as not to damage the surroundingnormal tissue, while treating a tumor (e.g., small breast cancer lesionsor ovarian tumors) at low temperature and release medication and Wntinhibitor or Rock inhibitors, such as Fasudil (HA-1077 a selectiveRhoA/Rho kinase (ROCK) inhibitor), Y-27632, small molecule inhibitor ofROCK1 and ROCK2 and low molecular weight heparin which act as ananti-inflammatory agent and inhibit Wnt activation, from thethermosensitive nanoparticle under the control of the temperature toprevent excessive inflammatory response in the treated organ and theincreased tumor biomarkers in the circulation after the thermotherapyhas an important diagnostic (i.e., indicating presence of a tumor) andtherapeutic value as biomarkers for the future management of thepatient.

In one embodiment, the thermal energy is applied to the body to find outif the antibody/medication coated pluralities of nanoparticles withactivatable cell-penetrating peptides (ACPPs) nanoshells with PFCL ornanocages, etc. are accumulated at a potential place, such as brain,breast, prostate, ovaries, etc., the nanoparticles when heated withthermal energy, such as AMF or focused low power ultrasound that can berecorded by a receiver and image the temperature, while recognizing thelocation of the potential tumor also in conjunction with MRI, or CT-scanor PET-scan, etc.

In one embodiment, the recoding or temperature imaging system isconnected to the thermal delivery unit via a processor or proportionalintegral derivative (PID) controller to prevent increase of thetemperature above predetermined level or maintaining or reducing thethermal energy or stopping it.

In one embodiment, the thermosensitive polymer of the nanoparticles ormicelles carries a dye, such as fluorescein, which is released at thetemperature of 41-43 degrees C. in the circulation and its presence inthe serum can be verified rapidly using a UV radiation indicating thatthe thermal energy created a temperature of 41-43 degrees C. at the siteof the nanoparticles.

In one embodiment, the functionalized pluralities of nanoparticles withactivatable cell-penetrating peptides (ACPPs) are coated withthermosensitive polymers conjugated with medication, monoclonalantibodies and checkpoint inhibitors, and are injected intravenously,intra-arterially, locally, systemically, subcutaneously,subconjunctival, orally, or by inhalation. After administration, thefunctionalized nanoparticles find the tumor cells expressing the tumorsurface antigen, attach to the cell membrane receptors of the tumorcells, during the thermotherapy at temperature of 41-43 degrees C. whenthe thermosensitive polymer melts, the medication, monoclonal antibodiesand checkpoint inhibitors and VLP, etc. from the thermosensitivepolymers are released precisely at the tumor site, eliminating thepresent practice of administrating the unbound medication, or unboundcheckpoint inhibitors in the circulation, which can kill the exposednormal cells by the medication or attack the cells of the body whichalso express normally checkpoint inhibitors, Rock inhibitors, such asFasudil, etc. to reduce inflammatory process and production of andTGF-β. The latter induces various side effects as autoimmune response tovarious organs of the patient, such as the lung, digestive tract, skin,heart or kidney etc. that are difficult to manage while eliminating thetumor with a precise and localized immune response.

In one embodiment, the functionalized or liposomes or nanoparticles arecoated with thermosensitive polymers, conjugated with a dye and with Wntinhibitors that reduces the post-treatment severe inflammatory responseand reduces the impulse for tumor cell multiplication by inactivation ofthe Wnt pathway, and the increased tumor biomarkers in the circulationafter the thermotherapy has an important diagnostic (i.e., indicatingpresence of a tumor) and therapeutic value as biomarkers for the futuremanagement of the patient.

In one embodiment, the functionalized pluralities of nanoparticles withactivatable cell-penetrating peptides (ACPPs) are conjugated with therock inhibitor Fasudil (HA-1077), a selective RhoA/Rho kinase (ROCK)inhibitor, or Y-27632, small molecule inhibitor of ROCK1 and ROCK2, andlow molecular weight heparin or IL-6 inhibitors etc. to dampenpostoperative cytokine inflammatory response and also block the Wntpathway inside the proliferating cancer cells or inflammatory cells seenafter monoclonal antibodies or chemotherapy.

In one embodiment, the functionalized pluralities of nanoparticles withactivatable cell-penetrating peptides (ACPPs) contain medications withthe rock inhibitor Fasudil (HA-1077), a selective RhoA/Rho kinase (ROCK)inhibitor, or Y-27632, small molecule inhibitor of ROCK1 and ROCK2, andlow molecular weight heparin or IL-6 inhibitors along with slow releasepolymers, such as polylactic or polyglycolic acid or combinations ofthem carrying immunosuppressive agents to reduce the cytokine responsein CAR-T cell immunotherapy.

In one embodiment, where there are tumor recurrences after initialgenetically modified CAR-T cell therapy in hematological malignanciessuch as lymphomas or leukemias or in solid tumors, such as melanoma,glioblastoma, ovarian, breast cancer, intestinal and colon cancer,prostate cancer, sarcoma, bone cancer, one administers intravenously,intraperitoneally, or subcutaneously, a combination of antibody coatednanoparticles conjugated with an antineoplastic medication along withWnt inhibitors or a Rock inhibitors, and low molecular weight heparin orIL-6 inhibitors, ipilimumab, nivolumab, avastin, or aflibercept, andapplies external thermal energy to heat up the functionalizedpluralities of nanoparticles with activatable cell-penetrating peptides(ACPPs) to release the medications at the temperature of 41-43 degreesC. or higher to inhibit Wnt activation by the metastatic cell and killthe tumor cells by increasing the nanoparticles temperature to 56degrees C. to kill the tumor cells and obtain the increased tumorbiomarkers from the circulation after the thermotherapy, to confirmdiagnosis the recurrent cancer and cold storage of the biomarkers forfuture management of the patient with recurrences or making a vaccinecombining the biomarkers, VLP, or oncolytic viruses, monoclonalantibodies, IL-2, bee toxins or other immune stimulators conjugated withantibody/medication polymeric coated nanoparticles and checkpointinhibitors such as PD-1, Jagged 1 inhibitor 15D11, for a lasting immunestimulation, etc., and Rock inhibitors or Wnt inhibitors, such asniclosamide, to be administered as needed, subcutaneously orsystemically to attach the nanoparticles with activatablecell-penetrating peptides (ACPPs) to the potential tumor cells, applyingthermal energy to release the medication from the thermosensitivepolymer coated nanoparticles and damage the tumor recurrence and toreactivate the immune response for a longer time up to months andeliminate the tumor cells and its potential metastatic lesions.

In one embodiment, where there are tumor recurrences after initialgenetically modified CAR-T cell therapy in hematological malignanciessuch as lymphomas or leukemias or in solid tumors, such as ocular orskin melanoma, glioblastoma, ovarian, breast cancer, intestinal andcolon cancer, prostate cancer, sarcoma, bone cancer, one administersintravenously, intraperitoneally, or subcutaneously, a combination ofantibody coated pluralities of nanoparticles conjugated withthermosensitive polymers and an antineoplastic medication along with Wntinhibitors or a Rock inhibitors, and low molecular weight heparin orIL-6 inhibitors, and ipilimumab, nivolumab, avastin, or aflibercept, ordiphenylpyrazole and application of external thermal energy to heat upthe functionalized pluralities of nanoparticles with activatablecell-penetrating peptides (ACPPs) to release the medications at thetemperature of 41-43 degrees C. or higher to inhibit Wnt activation bythe metastatic cell and kill the tumor cells by increasing thenanoparticles temperature to 56 degrees C. to kill the tumor cells andobtain the increased tumor biomarkers from the circulation after thethermotherapy, to confirm diagnosis the recurrent cancer and coldstorage of the biomarkers for future management of the patient withrecurrences or making a vaccine combining the biomarkers, VLP oroncolytic viruses, monoclonal antibodies, IL-2, bee toxins or otherimmune stimulators and checkpoint inhibitors conjugated withantibody/medication, an α-synuclein, diphenyl-pyrazole or compoundanle138b that blocks Aβ channels leading to dysregulation of autophagy,in advanced ocular or skin melanoma, coated nanoparticles to beadministered as needed, subcutaneously or systemically to attach thenanoparticles applying thermal energy to release the medication from thethermosensitive polymer coated pluralities of nanoparticles and damagethe tumor/melanoma recurrence and to reactivate the immune response andeliminate the tumor while antibody coated nanoparticles/checkpointinhibitors and VLP attach to the localized or circulating tumor cellsand their exosomes, which are carrying a checkpoint protein, such asPD-L1 (to disguise themselves), and the tumor cells are recognized bythe T cells, which together with killer cells phagocytose them, andenhance immune response to the tumor and its exosomes and thecirculating cells.

In one embodiment, ivermectin or niclosamide can be given orally at alow dose in conjunction with the standard immunothermotherapy procedure.

The Ras oncogenes and the proteins (Ras) encoded genes have had animportant roles in the pathogenesis of the human cancer affecting >50%of colon and >90% of pancreatic cancers. Ras proteins, aremembrane-bound GTP-binding proteins that serve as molecular switches inmutagenic signal transduction.

In one embodiment, inhibitors, such as Farnesyl or transferase, are usedto block Ras activation and other relevant pathway inhibitors of PK1 andPK8 and P13K, RAF/MEK/ERK, PI3K/AKT/mTOR and RalA/B pathways that can beinhibited by administering gemcitabine, wortmannin, LY294002 andtipifarnib alone or in combination with Gemcitabine or rapamycin andAZD8055(aTORKi), everolimus, and avastin, aflibercept and quenched dyeor another indicator conjugated with antibody/medication coatedthermosensitive polymers and/or pluralities of nanoparticles with ACPPintravenously, intra-arterially, in the cerebrospinal fluid, locally,intraperitoneally, and release the dye and the medication by applyingexternal, local, or internal thermal energy and damage the tumor cellsthat are damaged by the temperature of 41-43 degrees C. to 56 degreesC., thereby enhancing the effect of immunotherapy or medication andprevent production of medication resistance in these diseases, such aspancreatic cancer, breast cancer and brain cancer, includingglioblastoma, glioma, prostate cancer, ovarian cancer, and release theantigenic cell content in the circulation to incite an enhanced immuneresponse and obtaining new biomarkers from the blood to produce vaccineand administering them with checkpoint inhibitors, such as PD-1, PD-11,CTLA-4, Jagged 1 inhibitor 15D11, etc., and low molecular weight heparinor IL-6 inhibitors and Rock inhibitors, such as Fasudil, or Wntinhibitors, such as niclosamide.

In one embodiment, the pluralities of antibody/medication coatednanoparticles conjugated with either thermosensitive coated polymers ora combination of small liposomes containing functionalized pluralitiesof nanoparticles with activatable cell-penetrating peptides (ACPPs) thatact as thermal energy contrast agents are administered intravenously,intra-arterially, locally, intra-arterially, targeting multiple cancerssuch as leukemia, glioblastoma with cell surface GPC2 or glioma, orretinoblastoma or ovarian cancer or breast cancer, triple-negativebreast cancer cells, gastric cancer colon cancer, or melanomas, acutelymphocytic leukemia (ALL), etc. to image the lesion and the temperatureusing contrast agents, such as a liposome containing nanoshells, ornanocages containing, in addition, either a medication Wnt inhibitor,Avastin, aflibercept, ipilimumab nivolumab, or rock inhibitor inliposomal formulations, or buformin or phenformin, gemtuzumab or a dyeor an ultrasound contrast agent such as PFCL to create a popping soundwhen heated under the control of the thermal energy delivery unit toreach the boiling point of the PFCL at temperature of 56 degrees C.temperature recording it by a microphone indicating the 56 degrees C.temperature has been achieved and to damage to the tumor cell, and notheat up the tissue further to limit thermal damage by heat spillover tothe normal cells surrounding the tumor, and the increased tumorbiomarkers in the circulation after the thermotherapy has an importantdiagnostic value by proving the presence of a tumor and/or use thebiomarkers to make a vaccine for the future management of the patientrecurrences.

In one embodiment, the antibody/medication and a-synuclein coatednanoparticle is conjugated with ACPP and a member of specific bindingpair, a cellular receptor-agonist or antagonist pair, anenzyme-substrate pair, and/or an antibody-antigen pair, a virus, orvirus like particles (VLP) or oncolytic viruses, monoclonal antibodies,IL-2, bee toxins or other immune stimulators and Wnt inhibitor or Rockinhibitor with or without macrolides, such as cyclosporine A,mycophenolic acid, tacrolimus or ascomycin, is injected with monoclonalantibodies against the desired cancer along with a thermosensitivepolymer, such as chitosan, carrying an antineoplastic medication, etc.and unquenched fluorescein or bubble liposomes carrying fluoresceinwhich contain air pockets or nanoemulsions of PFC to be exposed tothermal energy releasing the medications at the temperature of 41-43degrees C. as evidenced by the release of the dye in the circulation toenhance the patient's immune response to the tumor such as glioblastoma,melanoma, ocular melanoma, metastatic melanoma, breast cancer, ovariancancer, etc. and induce an enhanced immune therapy.

In one embodiment, the antibody-coated nanoparticle is conjugated withACPP and a virus, or virus like particles (VLP), or oncolytic viruses,monoclonal antibodies, IL-2, bee toxins or other immune stimulatorsalong with checkpoint inhibitors, such as PD-1, PD-L1, CTLA-4, Jagged 1inhibitor 15D11, and Wnt inhibitors or Rock inhibitors, and lowmolecular weight heparin or IL-6 inhibitors and is injected with amonoclonal antibody against the desired cancer along with athermosensitive polymer, such as chitosan, carrying an antineoplasticmedication and unquenched fluorescein, or bubble liposomes carryingfluorescein which contain air pockets or nanoemulsions of PFC to beexposed to thermal energy releasing the medications, etc. at thetemperature of 41-43 degrees C. as evidenced by the release of the dyein the circulation to enhance the patient's immune response to the tumorsuch as glioblastoma, melanoma, ocular melanoma, metastatic melanoma,breast cancer, ovarian cancer, prostate, lung cancer, etc. along withenhanced immune therapy.

In one embodiment, the antibody coated nanoparticle is conjugated withACPP and with a member of a specific binding pair, such as astreptavidin-biotin pair, a cellular receptor-agonist or antagonistpair, an enzyme-substrate pair, and/or an antibody-antigen pair. Theantibody coated nanoparticle may be associated with a virus, e.g., amodified virus, Imlygic, Telomelysin, VCP inhibitor, or virus-likeparticles (VLP) made from modified plant viruses or oncolytic viruses,monoclonal antibodies, IL-2, bee toxins or other immune stimulatorsalong with checkpoint inhibitors, such as PD-1, PD-11, CTLA-4, Jagged 1inhibitor 15D11. VLP, plant viral capsids or membranes lacking thegenetic components of the virus and a Wnt inhibitor or Rock inhibitor,and low molecular weight heparin or IL6 inhibitors are injected withmonoclonal antibody/medication against the desired cancer along with athermosensitive polymer, such as chitosan, carrying an antineoplasticmedication and unquenched fluorescein, or bubble liposomes carryingfluorescein which contain air pockets or nanoemulsions of PFC to beexposed to thermal energy releasing the medications, etc. at thetemperature of 41-43 degrees C. as evidenced by the release of the dyein the circulation to enhance the patient's immune response to thetumor, such as glioblastoma, melanoma, ocular melanoma, metastaticmelanoma, breast cancer, ovarian cancer, prostate, lung cancer, etc.

In one embodiment, the antibody coated nanoparticle is conjugated withACPP and with a virus, e.g., a modified virus, a tumoricidal virus,and/or an adeno-associated virus (AAV) or virus like particles (VLP)made from modified plant viruses or oncolytic viruses, monoclonalantibodies, IL-2, bee toxins or other immune stimulators along withcheckpoint inhibitors, such as PD-1, PD-L1, CTLA-4, Jagged 1 inhibitor15D11. VLP, plant viral capsids or membranes lacking the geneticcomponents of the virus, and thus unable to multiply in the organism,and a Wnt inhibitor or Rock inhibitor is injected with monoclonalantibodies against the desired cancer along with a thermosensitivepolymer, such as chitosan, carrying an antineoplastic medication andunquenched fluorescein or bubble liposomes carrying medication(s) whichcontain air pockets or nanoemulsions of PFC to be exposed to thermalenergy releasing the medications etc. at the temperature of 41-43degrees as evidenced by the release of the medication locally or in thecirculation to enhance the patient's immune response to the tumor, suchas glioblastoma, melanoma, ocular melanoma, metastatic melanoma, breastcancer, ovarian cancer, prostate, lung cancer, etc.

In one embodiment, ivermectin nanoparticles conjugated withfunctionalized liposomes at pictogram concentrations given inside thetumor or intra-arterially causes an increase cell permeability thatmakes the tumor cells more susceptible to up take of chemotherapy, whileaddition of Rock inhibitors and diphenylpyrazole, and low molecularweight heparin or IL6 inhibitors reduce the side effects of inflammationin the immunotherapy and thermotherapy to prevent cytokine storm.

In one embodiment, ivermectin given as a non-toxic dose orally causes anincrease cell permeability of the tumor makes the tumor cell moresusceptible to up take of chemotherapy, while addition of Rockinhibitors, such as Fasudil, orally or intravenously anddiphenylpyrazole or and low molecular weight heparin or IL-6 inhibitors,reduce the side effects of inflammation in the immunotherapy andthermotherapy to prevent cytokine storm.

In one embodiment, rock inhibitors Fasudil (HA-1077 a selective RhoA/Rhokinase (ROCK) inhibitor, or Y-27632, small molecule inhibitor of ROCK1and ROCK2, and low molecular weight heparin or IL-6 inhibitors, etc. inliposomal preparation are administered systemically intravenously,intra-arterially locally, intra peritoneal, or in the cerebrospinalfluid with Biologic Response Modifiers using functionalized pluralitiesof nanoparticles with activatable cell-penetrating peptides (ACPPs)coated with rock inhibitors, Wnt inhibitors, Temozolomide, Cetuximab inthermosensitive polymers to release the medication at the desired placeat a desired time or combine them with standard anti-inflammatoryagents, etc., such as Steroids, Dexamethasone NASIDs, etc. and deliverpluralities of nanoparticles (i.e., biodegradable or non-biodegradablenanoparticles) administered systemically intravenously, intra-arteriallylocally, intra peritoneal or in the cerebrospinal fluid as thermalcontrast agents to prevent a cytokine storm in cancer immune therapy andremove cytokines after the therapy by electrophoresis, plasmapheresis orplasma exchange, or ARF6 inhibition, etc. to prevent the cytokine storm.

In another embodiment, monoclonal antibody therapies and other biologicsall are conjugated with the antibody/medication coated pluralities ofnanoparticles with activatable cell-penetrating peptides (ACPPs), otherbiologics include soluble receptors, cytokines, and natural cytokineantagonist administered in the thermosensitive polymeric coating of thefunctionalized nanoparticles/liposomes systemically, intravenously,locally, intra-arterially, or in a body cavity to target the tumor cellsprecisely to prevent the cytokine storm.

In another embodiment, humanized and chimeric antibodies areadministered via thermosensitive polymer coated antibody conjugatedpluralities of nanoparticles with activatable cell-penetrating peptides(ACPPs) intravenously or subcutaneously, and include TNF-alphainhibitors, anti-B lymphocyte therapy, IL-6 inhibitors, and lowmolecular weight heparin or cytotoxic T-lymphocyte-associated protein 4(CTLA-4) fusion inhibitors, and IL-2 receptor antagonists and targetlanthionine synthetase C-like 2 (LANCL2) for treating autoimmune diseaseadministered in the thermosensitive polymeric coating of thefunctionalized nanoparticle systemically, intravenously,intra-arterially, locally, intraperitoneally, or in the cerebrospinalfluid, and release them at a desired place with thermotherapy to preventa cytokine storm.

In one embodiment, Rock inhibitors, such as Fasudil (HA-1077 a selectiveRhoA/Rho kinase (ROCK) inhibitor, or Y-27632, small molecule inhibitorof ROCK1 and ROCK2 and low molecular weight heparin or IL-6 inhibitors,etc. with Infliximab are administered in a thermosensitive polymericcoating of the functionalized nanoparticle with activatablecell-penetrating peptides (ACPPs) systemically, intravenously,intra-arterially, locally, orally, intraperitoneally, or in thecerebrospinal fluid and released at a desired place with thermotherapyto prevent a cytokine storm.

In one embodiment, Rock inhibitors, such as Fasudil (HA-1077) aselective RhoA/Rho kinase (ROCK) inhibitor, or Y-27632, small moleculeinhibitor of ROCK1 and ROCK2, and low molecular weight heparin or IL-6inhibitors, etc. in liposomal preparation are administered systemically,intravenously, intra-arterially, locally, intraperitoneally, orally orin the cerebrospinal fluid, with an antibody coated plurality ofnanoparticles with activatable cell-penetrating peptides (ACPPs),conjugated with thermosensitive nanoparticles and Adalimumab which is ahumanized antibody administered subcutaneously, and released at adesired place with thermo-immunotherapy to prevent a cytokine storm andto enhance the effect of immunotherapy or medication by thermotherapyand prevent production of medication resistance in these cancers.

In one embodiment, Rock inhibitors, such as Fasudil (HA-1077), aselective RhoA/Rho kinase (ROCK) inhibitor, or Y-27632, a small moleculeinhibitor of ROCK1 and ROCK2, and low molecular weight heparin or IL-6inhibitors etc. in a functionalized liposomal preparation areadministered systemically intravenously, intra-arterially, locally,intraperitoneally, or in the cerebrospinal fluid, and they are releasedat a desired place with thermotherapy to prevent a cytokine storm and toenhance the effect of immunotherapy or medication by thermotherapy whilepreventing production of medication resistance in these cancers.

In one embodiment, Rock inhibitors, such as Fasudil (HA-1077), aselective RhoA/Rho kinase (ROCK) inhibitor, and low molecular weightheparin or IL-6 inhibitors or Y-27632, small molecule inhibitor of ROCK1and ROCK2 with antibody/medication coated pluralities of nanoparticlesconjugated with thermosensitive nanoparticles with activatablecell-penetrating peptides (ACPPs) and Golimumab, are delivered as asubcutaneous injection in cases that are refractory to treatment withcalcineurin inhibitors, corticosteroid combination therapy and releasethem at a desired place with thermo-immunotherapy to prevent a cytokinestorm.

In one embodiment, Rock inhibitors, such as Fasudil (HA-1077), aselective RhoA/Rho kinase (ROCK) inhibitor, or Y-27632, small moleculeinhibitor of ROCK1 and ROCK2, and low molecular weight heparin or IL-6inhibitors etc. are administered in a liposomal preparation containingquenched fluorescein or bubble liposomes carrying fluorescein whichcontain air pockets or nanoemulsions of PFC with antibody coatedpluralities of nanoparticles with activatable cell-penetrating peptides(ACPPs), conjugated with thermosensitive nanoparticles and Certolizumabpegol, dosing depends on the condition for which it is given, althoughin most cases it is given as a 400-mg weekly subcutaneous injection asneeded and released at a desired place with thermo-immunotherapy toprevent a cytokine storm.

In one embodiment, Rock inhibitors, such as Fasudil (HA-1077), aselective RhoA/Rho kinase (ROCK) inhibitor, or Y-27632, small moleculeinhibitor of ROCK1 and ROCK2, and low molecular weight heparin or IL6inhibitors etc., Avastin, aflibercept, ipilimumab, and/or nivolumab inliposomal preparation containing quenched fluorescein or another dye orindicator/medication or bubble liposomes carrying fluorescein/gene whichcontain air pockets or nanoemulsions of PFC are administered withantibody coated nanoparticles with activatable cell-penetrating peptides(ACPPs),conjugated with thermosensitive nanoparticles withcell-penetrating peptides (CPPs) and Rituximab, an anti-CD20 moleculegiven via intravenous infusion, to kill B lymphocytes, and thuseliminate them from the inflammatory process and release them at adesired place with thermo-immunotherapy to prevent a cytokine storm.

In one embodiment, Rock inhibitors in liposomal preparation containingfluorescein are administered systemically, intravenously,intra-arterially, locally, intraperitoneally, or in the cerebrospinalfluid, with antibody coated pluralities of nanoparticles withcell-penetrating peptides (CPPs), conjugated with thermosensitivenanoparticles and the IL-6 inhibitor tocilizumab and released at adesired place with thermo-immunotherapy to prevent a cytokine storm.

In one embodiment, Rock inhibitors, such as Botox or botulinum toxin atpicogram to one nanogram concentrations is given subcutaneously,locally, or less than one pictogram concentration with antibody coatedpluralities of nanoparticles with cell-penetrating peptides (CPPs),conjugated with thermosensitive nanoparticles and the IL-6 inhibitor,and low molecular weight heparin or tocilizumab, and released at adesired place with thermo-immunotherapy to prevent a cytokine storm.

In one embodiment, Rock inhibitors, such as Fasudil (HA-1077), aselective RhoA/Rho kinase (ROCK) inhibitor, or Y-27632, a small moleculeinhibitor of ROCK1 and ROCK2, etc. are administered systemicallyintravenously, intra-arterially, locally, intraperitoneally, or in thecerebrospinal fluid, in a liposomal preparation containing fluoresceinwith antibody coated pluralities of nanoparticles with cell-penetratingpeptides (CPPs), conjugated with a thermosensitive nanoparticles andwith the IL-2 receptor antagonist daclizumab and low molecular weightheparin or IL-6 inhibitors and they are released at a desired place withthermotherapy and temperature of 41-43 degrees C. to prevent a cytokinestorm, and prevent production of medication resistance in these cancers.

In one embodiment, Rock inhibitors, such as Botox, at pictogramconcentrations are administered with antibody coated pluralities ofnanoparticles with cell-penetrating peptides (CPPs), conjugated withthermosensitive nanoparticles and Alkylating agents and ipilimumab,nivolumab, administered systemically intravenously, intra-arteriallylocally, intraperitoneally, and low molecular weight heparin or IL-6inhibitors or in the cerebrospinal fluid, and they are released at adesired place with thermotherapy at the temperature of 41-43 degrees C.to prevent a cytokine storm.

In one embodiment, Rock inhibitors, such a Fasudil (HA-1077), aselective RhoA/Rho kinase (ROCK) inhibitor, or Y-27632, small moleculeinhibitor of ROCK1 and ROCK2, and low molecular weight heparin or IL6inhibitors, etc. in a liposomal preparation containing fluorescein areadministered with antibody coated pluralities of nanoparticles withcell-penetrating peptides (CPPs), conjugated with thermosensitivenanoparticles and/or small molecules or Canakinumab is approved for usein juvenile idiopathic arthritis administered systemically,intravenously, intra-arterially, locally, intraperitoneally, or in thecerebrospinal fluid, and they are released at a desired place withthermotherapy at the temperature of 41-43 degrees C. to reduceinflammatory processes while prevent production of medication resistancein these cancers.

In one embodiment, Rock inhibitors, and low molecular weight heparin orIL-6 inhibitors are administered systemically, intravenously,intra-arterially, locally, intraperitoneally, or in the cerebrospinalfluid, with antibody coated pluralities of nanoparticles withcell-penetrating peptides (CPPs), conjugated with thermosensitivenanoparticles and Gevokizumab with or without small molecule Wntinhibitor and they are released at a desired place with thermotherapyand a temperature of 41-43 degrees C.

In one embodiment, Rock inhibitors are administered systemically,intravenously, intra-arterially, locally, intraperitoneally, or in thecerebrospinal fluid, with antibody coated pluralities of nanoparticleswith cell-penetrating peptides (CPPs), conjugated with thermosensitivenanoparticles and Gevokizumab with or without small molecule Wntinhibitor, and they are released at a desired place with thermotherapyand a temperature of 41-43 degrees C. or the Rock inhibitors or Wntinhibitors are given orally at a non-toxic dose once or can be repeatedmonthly if needed.

In one embodiment, Rock inhibitors, such as Fasudil (HA-1077), aselective RhoA/Rho kinase (ROCK) inhibitor, or Y-27632, a small moleculeinhibitor of ROCK1 and ROCK2, and low molecular weight heparin or IL-6inhibitors, etc. in a liposomal preparation containing medication(s) areadministered with antibody coated nanoparticles with cell-penetratingpeptides (CPPs), conjugated with thermosensitive nanoparticles andAlemtuzumab, which has been shown to be beneficial for use in B-cellchronic lymphocytic leukemia, and they are released at a desired placewith thermo-immunotherapy at the temperature of 41-43 degrees C. withlocal thermotherapy to reduce inflammatory processes while preventingproduction of medication resistance in these cancers because the cancercells do not survive the hyperthermia.

In one embodiment, Rock inhibitors are administered systemically,intravenously, intra-arterially, locally, intraperitoneally, or in thecerebrospinal fluid with antibody coated nanoparticles, conjugated withthermosensitive nanoparticles with cell-penetrating peptides (CPPs) andwith Sarilumab a monoclonal antibody that targets IL-6, and lowmolecular weight heparin preventing T- and B-cell activation anddifferentiation and releases them at a desired place with thermotherapyat the temperature of 41-43 degrees C. to reduce inflammatory processes.

In one embodiment, Rock inhibitors, and low molecular weight heparin orIL-6 inhibitors are administered systemically intravenously,intra-arterially locally, intraperitoneally, or in the cerebrospinalfluid with antibody/medication coated pluralities of nanoparticles,conjugated with thermosensitive nanoparticles with cell-penetratingpeptides (CPPs) and Sirolimus and release them at a desired place withthermotherapy at the temperature of 41-43 degrees C. to reduceinflammatory processes while preventing production of medicationresistance in these cancers because the cancer cells do not survive thehyperthermia.

In one embodiment, radiolabeled antibodies, such as Ibritumomabtiuxetan, is conjugated with a thermosensitive polymer, such aschitosan, coating antibody coated nanoparticle/liposomes to deliver themedication and low molecular weight heparin or IL-6 inhibitors, to thetumor cell systemically intravenously, intra-arterially locally,intraperitoneally, or in the cerebrospinal fluid and release them at adesired place with thermotherapy at the temperature of 41-43 degrees C.to reduce side effect of tiuxetan and Ibritumomab.

In one embodiment, the thermosensitive polymer of theantibody/medication coated nanoparticles with ACPP, containing aradiolabeled medication and low molecular weight heparin or IL-6inhibitors, are administered systemically intravenously,intra-arterially locally, intraperitoneally, or in the cerebrospinalfluid to damage the tumor cells as a combination thermoradiotherapy andreduce their side effect while preventing production of medicationresistance in these cancers because the cancer cells do not survive thehyperthermia.

In one embodiment, the thermosensitive polymer of theantibody/medication coated pluralities of nanoparticles, such as goldnanoparticles that enhance the effect of external x-ray or particle beamradiation contains mebendazole, taxol derivatives, an anti-CCR2 antibodyresults in blockade of radiation-induced monocytic myeloid-derivedsuppressor cells infiltration that create radiation resistance, andfibrous tissue scars, in addition Rock inhibitors, such as Fasudil etc,and low molecular weight heparin or IL-6 inhibitors, intra-arteriallylocally, in the body cavity given to reduce inflammatory process andscar tissue, and quenched fluorescein or another indicator with Wntinhibitors administered systemically intravenously, intra-arteriallylocally, intraperitoneally, or in the cerebrospinal fluid released withcontrolled thermotherapy when administered medication to damage thetumor cells as a combination thermoradiotherapy in glioblastoma,medulloblastoma, retinoblastoma, melanoma or other brain tumors, lungcancer acting on the microtubes of the cancer cells while preventingproduction of medication resistance in these cancers because the cancercells do not survive the hyperthermia, etc.

In one embodiment, the thermosensitive polymer of theantibody/medication coated nanoparticles contains mebendazole, taxolderivatives, and quenched fluorescein or another indicator with Rockinhibitors, and low molecular weight heparin or IL-6 inhibitors,checkpoint inhibitors administered systemically, intravenously,intra-arterially locally, intraperitoneally, or in the cerebrospinalfluid released with controlled thermotherapy when administeredmedication to damage the tumor cells as a combination thermoradiotherapyin glioblastoma, medulloblastoma, retinoblastoma, melanoma or otherbrain tumors, lung cancer acting on the microtubes of the cancer cellswhile preventing production of medication resistance in these cancersbecause the cancer cells do not survive the hyperthermia, etc.

In one embodiment, one or multiple agents are used to treat a number ofcancers alone with a monoclonal antibody conjugated with thermosensitiveantibody coated pluralities of nanoparticles with cell-penetratingpeptides (CPPs), nanoshell or nanocage treatment with temperaturesensitive polymers conjugated with the nanoparticles having ipilimumab,nivolumab, and/or temozolomide combined a small molecule Wnt inhibitor,ivermectin, or niclosamide along with paclitaxel, or mebendazole, VLP oroncolytic viruses, monoclonal antibodies, IL-2, bee toxins or otherimmune stimulators along with checkpoint inhibitors, such as PD-1,PD-11, CTLA-4, Jagged 1 inhibitor 15D11 administered intravenously orintra-arterially by injection, systemically intravenously, locally,intraocularly, in the tumor, intraperitoneally or in the cerebrospinalfluid at nanogram doses to be released from the antibody/medicationcoated nanoparticle at the tumor site using thermotherapy with eitheralternating magnetic field or focused low power ultrasound, a laser andsimultaneous release of a dye such as quenched fluorescein orindocyanine green from the nanoparticle's thermosensitive polymer ormagnetic nanoshells containing fluorescein and PFH under observationwith MRI or focused ultrasonic units, a CT-scan, or PET-scan, andrelease the medication at a desired place with thermotherapy at thetemperature of 41-43 degrees C. or higher up to 60 or more degrees C. asrecorded by a microphone or an ultrasonic receiver, and imaged with athermoacoustic imaging system for control of the temperature and toinduce an enhanced immunotherapy and to damage the tumor cells whileverifying the medication/dye release from the blood samples and obtainnewly released biomarkers from the tumor cells in the circulation tomake a vaccine with VLP and/or IL-2 combined with antibody coatednanoparticles.

In one embodiment, the focused low power ultrasound is combined with thealternating magnetic field to simultaneously heat and image the tissuewhile heating and killing the tumor at a lower time duration, thepluralities of nanoparticles with cell-penetrating peptides (CPPs) arecoated with thermosensitive polymers for drug release at temp. of 41-43degrees C., imaged with a thermoacoustic imaging system for control ofthe temperature and the popping sound of the cavitation sound is heardat the temperature of 56 degrees C. killing the tumor cells releasingtheir antigenic content to stimulate immune response to remove all thetumor cells, thereby eliminating the need for cellular immune therapy,and the increased tumor biomarkers in the circulation after thethermotherapy has an important diagnostic (i.e., indicating presence ofa tumor) and therapeutic value as biomarkers for the future managementof the patient.

In one embodiment, the focused ultrasound can be directed by a computercontrolled head to the exact location and borders of the tumor mass,which may be marked by focused high power ultrasound to heat up andcreate bubbles after heating the nanoshell or nanocage filled with PFCLhexane to its transition temperature of 40-43 C or higher degrees C.that can be recorded by an additional ultrasound receiver or amicrophone as a cavitation sound creating a precise ablation of a tumorunder ultrasonic observation and drug Wnt inhibitors and a Rho inhibitoror mebendazole, diphenylpyrazole, and low molecular weight heparin orIL-6 inhibitors release to kill the tumor while dampening thepost-therapy inflammation, thus enhancing the person's immune responsecells eliminating the need for cellular immune therapy, and theincreased tumor biomarkers in the circulation after the thermotherapyhas an important diagnostic (i.e., indicating presence of a tumor) andtherapeutic value as biomarkers for the future management of the patienthaving glioblastoma, medulloblastoma, ovarian tumor, or breast cancer,retinoblastoma, glioma, etc.

In one embodiment, a known Wnt inhibitor, such as ivermectin ormebendazole, conjugated with ipilimumab, nivolumab, and thermosensitivepolymer antibody coated nanoparticles, nanocages, nanoshells having thetemperature markers fluorescein and PFCL intravenously, intra-arteriallylocally, or in the cerebrospinal fluid systemically intravenously,locally, intraperitoneally to attach to the tumor receptors and heatedwith either an alternating magnetic field, or ultrasound or microwave toheat up the tumors and release the medication under control of thetemperature or 41-43 degrees C., then 56 degrees C., imaged with athermoacoustic imaging system for control of the temperature and aprocessor to release the medication and kill the tumor simultaneously bythermal energy releasing more biomarkers from the tumor to enhanceimmune response cells by releasing increased tumor biomarkers in thecirculation and eliminating the need for cellular immune therapy, andthe increased tumor biomarkers in the circulation after thethermotherapy has an important diagnostic (i.e., indicating presence ofa tumor) and therapeutic value as biomarkers for vaccine production forthe future management of the patient having glioblastoma,medulloblastoma, ovarian tumor, or breast cancer, retinoblastoma,gliomas, and administering them along with checkpoint inhibitors, suchas PD-1, Jagged 1 inhibitor 15D11, etc., and Rock inhibitors, and lowmolecular weight heparin or IL-6 inhibitors, or Wnt inhibitors, such asniclosamide etc.

In one embodiment, a known Wnt inhibitor, such as ivermectin and taxolor other medication, conjugated with thermosensitive polymerantibody/medication coated nanoparticles, nanocages, nanoshells havingthe temperature markers quenched fluorescein or another indicator andPFCL intravenously, intra-arterially locally, or in the cerebrospinalfluid to attach to the tumor receptors and heated with either analternating magnetic field, or focused low power ultrasound or microwaveto heat up the tumors and release the medication under control of thetemperature and a processor connected to thermal energy delivery systemand thermal imaging unit to release the medication and kill the tumorsimultaneously at a defined thermal energy temperature, and theincreased tumor biomarkers in the circulation after the thermotherapyhas an important diagnostic (i.e., indicating presence of a tumor) andtherapeutic value as biomarkers for the future management of the patientand production of vaccine for the patient's tumor and administering itwith checkpoint inhibitors, such as PD-1, Jagged 1 inhibitor 15D11,etc., and Rock inhibitors, such as Fasudil, or Wnt inhibitors, such asniclosamide and low molecular weight heparin or IL-6 inhibitors.

In one embodiment, a small molecule Wnt inhibitor and taxane areconjugated with a thermosensitive polymer having ipilimumab and/ornivolumab, antibody coated nanoparticles, nanocages, or nanoshellshaving the temperature markers quenched fluorescein and PFCLintravenously, intra-arterially locally, in the bladder, or in thecerebrospinal fluid to attach to the tumor receptors and heated witheither an alternating magnetic field, low power ultrasound, ormicrowaves to heat up the tumors and release the medication undercontrol of the temperature and a processor to release the medication andkill the tumor simultaneously by thermal energy and the increased tumorbiomarkers in the circulation after the thermotherapy has an importantdiagnostic (i.e., indicating presence of a tumor) and therapeutic valueas biomarkers for the future of the patient and production of vaccinefor the patient's tumor and administering it with checkpoint inhibitorssuch as PD-1, Jagged 1 inhibitor 15D11, etc., and Rock inhibitors, suchas Fasudil, or Wnt inhibitors, such as niclosamide, and low molecularweight heparin or IL-6 inhibitors.

In one embodiment, a known Wnt inhibitor, Ivermectin PEGylated lipidnanoemulsion conjugated along with the Rock inhibitor Fasudil (HA-1077),a selective RhoA/Rho kinase (ROCK) inhibitor, or Y-27632, a smallmolecule inhibitor of ROCK1 and ROCK2, etc. and decitabine withthermosensitive polymer antibody coated nanoparticles, nanocages, ornanoshells having the temperature markers fluorescein and PFCLintravenously, intra-arterially locally, or in the cerebrospinal fluidto attach to the tumor receptors and heated with either an alternatingmagnetic field, or low power ultrasound or microwave or radiofrequencyto heat up the tumors and release the medication under control of thetemperature and a processor to release the medication and kill the tumorsimultaneously by thermal energy 43 degrees C., imaged with athermoacoustic imaging system for control of the temperature to reducethe production of inflammatory cytokines by macrophages and theincreased tumor biomarkers in the circulation after the thermotherapyhas an important diagnostic (i.e., indicating presence of a tumor) andtherapeutic value as biomarkers for the future management of the patientand production of vaccine for the patient's tumor and administering itwith VLP and checkpoint inhibitors, such as PD-1, Jagged 1 inhibitor15D11, etc., and Rock inhibitors, such as Fasudil, or Wnt inhibitors,such as niclosamide to enhance immunotherapy, while Rock inhibitors andWnt inhibtors reduce TGF-β production after therapy and low molecularweight heparin or IL-6 inhibitors to reduce inflammation and scarformation.

In one embodiment, the oral dose for ivermectin is about 40-150microgram/kg, once in 7 days and the systemic dose is (10-100 ng/ml) in1-5 (ml) in physiologic solution, or more doses as needed, and the oraldose of Fasudil is 40 mg-80 mg and the systemic dose is 50 nanograms toa few micrograms.

In one embodiment, a small molecule Wnt inhibitor PKF118-310, theWnt/β-catenin pathway inhibitor and fasudil, a rock inhibitor Fasudil(HA-1077), a selective RhoA/Rho kinase (ROCK) inhibitor, or Y-27632, asmall molecule inhibitor of ROCK1 and ROCK2, and low molecular weightheparin or IL6 inhibitors, etc. are prepared as PEGylated lipidnanoemulsion conjugated and a rock inhibitor and metformin, buformin, orphenformin and syrosingopine to inhibit glucose metabolism in the tumorcells and conjugated with a thermosensitive polymer antibody/medicationcoated nanoparticles, nanocages, or nanoshells having the temperaturemarkers quenched fluorescein or another indicator and PFCL administeredintravenously, intra-arterially locally, or in the cerebrospinal fluidto attach to the tumor receptors and heated with either an alternatingmagnetic field, low power ultrasound, or microwaves to heat up thetumors and release the medication under control of the temperature,imaged with a thermoacoustic imaging system for control of thetemperature and a processor to release the medication and kill the tumorcells and starve those that might escape treatment preventing theirimportant glucose metabolism.

In one embodiment, a known Wnt inhibitor Ivermectin and doxorubicin(Adriamycin) or taxol derivatives are conjugated with thermosensitivepolymer antibody coated nanoparticles, nanocages, or nanoshells havingthe temperature markers quenched fluorescein or another indicator andPFCL conjugated with Niclosamide, Rock inhibitor Fasudil (HA-1077), aselective RhoA/Rho kinase (ROCK) inhibitor, or Y-27632, a small moleculeinhibitor of ROCK1 and ROCK2 and femformin, etc. in liposomalpreparation containing fluorescein, metformin, or buformin, andantineoplastic medication or monoclonal antibody rituximabintravenously, intra-arterially locally, or in the cerebrospinal fluidto attach to the tumor receptors and heated with either an alternatingmagnetic field, low power ultrasound, or microwaves to heat up thetumors and release the medication under control of the temperature witha thermoacoustic imaging system for control of the temperature initiallyand a processor to control the temperature, imaged at 41-43 degrees C.with the release of quenched fluorescein to release the medication andkill the tumor simultaneously by thermal energy subsequently at 50-56degrees C. with the low power focused ultrasound receiver showing theultrasound cavitation at a boiling point of PFCL in triple negativebreast cancer or in ovarian cancer, glioblastoma, melanoma,retinoblastoma, gastric cancer, etc. and the increased tumor biomarkersin the circulation after the thermotherapy has an important diagnosticvalue (i.e., confirming the presence of a tumor) and therapeutic valueas biomarkers for the future management of the patient with recurrencesby making a vaccine using the biomarkers and VLP or oncolytic viruses,monoclonal antibodies, IL-2, bee toxins or other immune stimulatorsconjugated with antibody coated nanoparticles to attach to the potentialtumor cells and administering it with checkpoint inhibitors, such asPD-1, Jagged 1 inhibitor 15D11, etc., and Rock inhibitors, such asFasudil, or Wnt inhibitors, such as niclosamide, while applying thermalenergy to damage recurrence of the tumor and induce reactivation of theimmune response to eliminate them and prevent the tumor's importantglucose metabolism with metformin, buformin, while antibody coatednanoparticles/checkpoint inhibitors and VLP attach to the localized orcirculating tumor cells and their exosomes, which are carrying acheckpoint protein, such as PD-L1 (to disguise themselves), and thetumor cells are recognized by the T cells, which together with killercells phagocytose them, and enhance the immune response to the tumor andits exosomes and the circulating cells.

In one embodiment, a known Wnt inhibitor, ivermectin and doxorubicin(Adriamycin) or taxol derivatives are conjugated with thermosensitivepolymer antibody coated nanoparticles, liposomes, micelles,nanocages, ornanoshells having the temperature markers quenched fluorescein oranother dye or indicator and PFCL conjugated with Niclosamide, Rockinhibitor Fasudil (HA-1077), a selective RhoA/Rho kinase (ROCK)inhibitor, or Y-27632, a small molecule inhibitor of ROCK1 and ROCK2 andfemformin, etc. in liposomal preparation containing fluorescein,metformin, or buformin, or metformin and with or without syrosingopine,cinnamaldehyde and an antineoplastic medication or monoclonal antibodyrituximab intravenously, intra-arterially locally, or in thecerebrospinal fluid to attach to the tumor receptors and heated witheither an alternating magnetic field, ultrasound, or microwaves to heatup the tumors and release the medication under control of thetemperature, imaged with a thermoacoustic imaging system for control ofthe temperature and a processor to control the temperature initially at41-43 degrees C. with the release of the medication and kill the tumorsimultaneously by thermal energy and its control with CBE and hCBE, orRF generated by the heated tissue in triple negative breast cancer or inovarian cancer, glioblastoma, melanoma, retinoblastoma, gastric cancer,etc. and the increased tumor biomarkers in the circulation after thethermotherapy has an important diagnostic value (i.e., confirming thepresence of a tumor) and therapeutic value as biomarkers for the futuremanagement of the patient with recurrences by making a vaccine using thebiomarkers and VLP or oncolytic viruses conjugated with antibody coatednanoparticles and administering it with checkpoint inhibitors, such asPD-1, Jagged 1 inhibitor 15D11, etc., and Rock inhibitors, such Fasudil,or Wnt inhibitors, such as niclosamide, to attach to the potential tumorcells while applying thermal energy to damage recurrence of the tumorand induce reactivation of the immune response to eliminate them andprevent the tumor's important glucose metabolism with metformin,buformin and syrosingopine or cinnamaldehyde.

In one embodiment, a known Wnt inhibitor, ivermectin, niclosamide,metformin, or buformin, are conjugated with thermosensitive polymerantibody/medication coated nanoparticles, nanocages, nanoshells havingthe temperature markers quenched fluorescein or another indicator andPFCL intravenously, intra-arterially locally, or in the cerebrospinalfluid to attach to the tumor receptors and heated with either analternating magnetic field, high power ultrasound, or microwaves to heatup the tumors and release the medication under control of thetemperature of 41-43 degrees C. or 50-56 degrees C., imaged with athermoacoustic imaging system for control of the temperature and aprocessor to release the medication and kill the tumor simultaneously bythermal energy in treatment of glioblastoma or glioma, or ovarian canceror breast cancer and the increased tumor biomarkers in the circulationafter the thermotherapy has an important diagnostic (i.e. indicatingpresence of a tumor) and therapeutic value as biomarkers for the futuremanagement of the patient and eliminating the need for modified CAR-Tadministration to the patient.

In one embodiment, a known Wnt inhibitor, niclosamide and taxol, ordaunorubicin, ara-C or cytarabine or metformin, buformin or phenforminwith or without syrosingopine are conjugated with a thermosensitivepolymer antibody coated liposomes, micelles, nanoparticles and ACPP,nanocages or nanoshells having the temperature markers quenchedfluorescein and PFCL intravenously, intra-arterially, locally in thetumor, or in the cerebrospinal fluid to attach to the tumor receptorsand heated with either an alternating magnetic field, focusedultrasound, or microwaves to heat up the tumors and release themedication under control of the temperature and a processor to releasethe medication and kill the tumor simultaneously by localized thermalenergy in treatment of glioblastoma or glioma, or ovarian cancer orbreast cancer, gastric cancer, colon cancer, or melanomas, Mantle celllymphoma preferentially killing the cancer stem cells, etc. and theincreased tumor biomarkers in the circulation after the thermotherapyhas an important diagnostic (i.e., indicating presence of a tumor) andtherapeutic value as biomarkers for vaccine production with or withoutVLP attached to antibody coated nanoparticles and administering it withcheckpoint inhibitors such as PD-1, Jagged 1 inhibitor 15D11, etc., andRock inhibitors, such as Fasudil, or Wnt inhibitors, such asniclosamide, with cell-penetrating peptides (CPPs) for the management ofthe future recurrences in the patient and eliminating the need forgenetically CAR-T administration to the patient, and preventing theirimportant glucose metabolism with metformin, buformin or phenforminwhile antibody coated nanoparticles/checkpoint inhibitors and VLP attachto the localized or circulating tumor cells and their exosomes, whichare carrying a checkpoint protein, such as PD-L1 (to disguisethemselves), and the tumor cells are recognized by the T cells, whichtogether with killer cells phagocytose them, and enhance the immuneresponse to the tumor and its exosomes and the circulating cells.

In one embodiment, a known Wnt inhibitor, niclosamide and taxol, ordaunorubicin, ara-C or cytarabine or metformin, buformin or phenforminwith or without syrosingopine are conjugated with a thermosensitivepolymer antibody/medication coated nanoparticles and ACPP, nanocages ornanoshells having the temperature markers quenched fluorescein and PFCLintravenously, intra-arterially locally, or in the cerebrospinal fluidto attach to the tumor receptors and heated with either an alternatingmagnetic field, low power focused ultrasound, or microwaves to heat upthe tumors and release the medication under control of the temperatureand a processor to release the medication and kill the tumorsimultaneously by thermal energy, imaged with a thermoacoustic orultrasound CBE or in combination with HCBE imaging system for control ofthe temperature in treatment of glioblastoma or glioma, or ovariancancer or breast cancer, gastric cancer, colon cancer, or melanomas,Mantle cell lymphoma preferentially killing the cancer stem cells, etc.and the increased tumor biomarkers in the circulation after thethermotherapy has an important diagnostic (i.e., indicating presence ofa tumor) and therapeutic value as biomarkers for vaccine production withor without VLP or oncolytic viruses attached to antibody coatednanoparticles and administering them with checkpoint inhibitors, such asPD-1, Jagged 1 inhibitor 15D11, etc., and Rock inhibitors, such asFasudil or Wnt inhibitors, such as niclosamide, with cell-penetratingpeptides (CPPs) for the management of the future recurrences in thepatient and eliminating the need for genetically modified CAR-Tadministration to the patient, and preventing their important glucosemetabolism with metformin, buformin or phenformin and syrosingopine.

In one embodiment, a known Wnt inhibitor, Niclosamide and VLP, andcheckpoint inhibitors and metformin, or buformin are conjugated with athermosensitive polymer antibody coated nanoparticles with ACPP,nanocages, or nanoshells having the temperature markers quenchedfluorescein or another indicator and PFCL intravenously,intra-arterially locally, or in the cerebrospinal fluid to attach to thetumor receptors and heated with either an alternating magnetic field,high power ultrasound, or microwaves to heat up the tumors and releasethe medication under control of the temperature of 42 to 43 degrees C.and, imaged with a thermoacoustic/ultrasound imaging system for controlof the temperature and a processor to release the medication and killthe tumor simultaneously by thermal energy in treatment of glioblastomaor glioma, or ovarian cancer or breast cancer and the increased tumorbiomarkers in the circulation after the thermotherapy has an importantdiagnostic (i.e. indicating presence of a tumor) and therapeutic valueas biomarkers for vaccine production with or without VLP or oncolyticviruses attached to antibody coated nanoparticles, and administeringthem with checkpoint inhibitors, such as PD-1, Jagged 1 inhibitor 15D11,etc., and Rock inhibitors, such as Fasudil, or Wnt inhibitors, such asniclosamide, with cell-penetrating peptides (CPPs) for the management ofthe future recurrences in the patient and eliminating the need forgenetically modified CAR-T administration to the patient and preventingtheir important glucose metabolism at the tumor site and enhanceimmunotherapy.

In one embodiment, the Wnt inhibitor, ivermectin, may be givenseparately orally or systemically at low concentration of 1-90nanograms/ml with thermotherapy of the glioblastoma, breast, ovarian,prostate cancer, lymphoma, leukemia along with thermotherapy usingantibody/medication coated nanoparticles, nanocages, or nanoshellshaving the temperature markers quenched fluorescein and PFCLintravenously, intra-arterially locally, intra-arterially locally, inthe body cavity such as bladder, mouth, nasal cavity, or in thecerebrospinal fluid to attach to the tumor receptors and heated witheither an alternating magnetic field, low power ultrasound, ormicrowaves to heat up the tumors and release the medication undercontrol of the temperature, imaged with a thermoacoustic ultrasound orultrasound CBE or in combination with HCBE imaging system for control ofthe temperature, and a processor to release the medication and kill thetumor simultaneously by thermal energy in treatment of glioblastoma orglioma, or ovarian cancer or breast cancer and the increased tumorbiomarkers in the circulation after the thermotherapy has an importantdiagnostic (i.e., indicating presence of a tumor) and therapeutic valueas biomarkers for vaccine production with or without VLP attached toantibody coated nanoparticles, and administering them with VLP,checkpoint inhibitors, such as PD-1, Jagged 1 inhibitor 15D11, etc., andRock inhibitors, such as Fasudil, or Wnt inhibitors, such asniclosamide, with cell-penetrating peptides (CPPs) for the management ofthe future recurrences in the patient and eliminating the need forgenetically modified CAR-T administration to the patient.

In one embodiment, a known Wnt inhibitor, niclosamide, and rituximab areconjugated with thermosensitive polymer antibody/medication coatednanoparticles, nanocages, or liposomes, nanoshells having thetemperature markers quenched fluorescein and PFCL intravenously, andcheckpoint inhibitors, Rock inhibitors, such as Fasudil, or ultrasoundCBE or in combination with HCBE imaging system, etc., intra-arteriallylocally, in the body cavity, such as in the bladder, or in thecerebrospinal fluid to attach to the tumor receptors and heated witheither an alternating magnetic field, focused ultrasound, or microwavesto heat up the tumors and release the medication under control of thetemperature, imaged with a thermoacoustic imaging system for control ofthe temperature and a processor to release the medication and kill thetumor simultaneously by thermal energy in treatment of glioblastoma orglioma, or ovarian cancer or breast cancer, gastric cancer colon cancer,or melanomas, bladder or prostate cancer, etc.

In one embodiment, a known Wnt inhibitor Niclosamide and rituximab areconjugated with thermosensitive polymer antibody/medication coatednanoparticles, nanocages, or nanoshells having the temperature markersquenched fluorescein and PFCL intravenously, and checkpoint inhibitors,but Rock inhibitors, such as Fasudil, etc., given orally or locally,intra-arterially locally, in the body cavity, such as in the bladder, orin the cerebrospinal fluid to attach to the tumor receptors and heatedwith either an alternating magnetic field, high power focusedultrasound, or microwaves to heat up the tumors and release themedication under control of the temperature and a processor to releasethe medication and kill the tumor simultaneously by thermal energy intreatment of glioblastoma or glioma, or ovarian cancer or breast cancer,gastric cancer colon cancer, or melanomas, bladder or prostate cancer,etc.

In one embodiment, a known Wnt inhibitor is conjugated with rockinhibitor Fasudil (HA-1077), a selective RhoA/Rho kinase (ROCK)inhibitor, or Y-27632, a small molecule inhibitor of ROCK1 and ROCK2,and low molecular weight heparin or IL-6 inhibitors, etc. in a liposomalpreparation containing fluorescein or metformin, buformin, or phenforminand thermosensitive polymer antibody coated nanoparticles, nanocages, ornanoshells having the temperature markers quenched fluorescein or otherdyes or indicators and PFCL intravenously, intra-arterially locally, orin the cerebrospinal fluid to attach to the tumor receptors and heatedwith either an alternating magnetic field, low power ultrasound, ormicrowaves, or laser of various wave length and duration from femtosecond to one second pulses to heat up the tumors and release themedication under control of the temperature, imaged with aphotoacoustic, thermoacoustic, ultrasound imaging system for control ofthe temperature and a processor to release the medication targetingmultiple signaling pathways (NF-κB, Wnt/β-catenin, Notch, ROS, mTORC1,and Stat3 and kill the tumors simultaneously by thermal energy in thetreatment of glioblastoma or glioma, or ovarian cancer or breast cancer,gastric cancer, colon cancer, or melanomas etc. and the increased tumorbiomarkers in the circulation after the thermotherapy has an importantdiagnostic (i.e. indicating presence of a tumor) and therapeutic valueas biomarkers in vaccine production along with antibody coatednanoparticles conjugated with VLP, and administering them withcheckpoint inhibitors, such as PD-1, Jagged 1 inhibitor 15D11, etc. forenhanced localized immunotherapy and low molecular weight heparin orIL-6 inhibitors, Rock inhibitors, such as Fasudil or Wnt inhibitors,such as niclosamide, for the future management of the recurrences inpatients and reduce the cytokine storm.

In one embodiment, ivermectin or a small molecule Wnt inhibitorPKF118-310 or Curcumin, and doxycycline derivatives, or vitamin D areconjugated with thermosensitive polymer antibody/medication coatednanoparticles with CPP, nanocages, or nanoshells having the temperaturemarkers quenched fluorescein or other dyes or indicators and PFCLintravenously, intra-arterially, locally, or in the cerebrospinal fluidto attach to the tumor receptors and heated with either an alternatingmagnetic field, high power ultrasound, or microwaves to heat up thetumors and release the medication under control of the temperature 43degrees C. to 50-56 degrees C. temperature, imaged with a thermoacousticimaging system for control of the temperature and a processor to releasethe medication and kill the tumor simultaneously by thermal energy intreatment of glioblastoma or glioma, or ovarian cancer, colon cancer,prostate cancer, or breast cancer, mammary stem cells cancers and theincreased tumor biomarkers in the circulation after the thermotherapyhas an important diagnostic (i.e. indicating presence of a tumor) andtherapeutic value as biomarkers for the future management of the patientremoving the cytokine from the plasma to avoid autoantibody and lowmolecular weight heparin or IL-6 inhibitors reduce the cytokine storm bythe use of electrophoresis, plasmapheresis, or plasma exchange or ARF6inhibition.

In one embodiment, in cytokine storm, with elevated levels of tumornecrosis factor (TNF) α, interleukin-6, granulocyte colony-stimulatingfactor, interleukin-1β, and interleukin-17 one administers systemictocilizumab at a dose of 8 mg per kilogram and or Intravenous immuneglobulin (IVIG)2 g per kilogram, or nanoparticles coated withthermosensitive polymers carrying, Rock inhibitors, and low molecularweight heparin or IL6 inhibitors mycophenolic acid, cyclosporine A, orother macrolides, infliximab, an anti-TNF-α antibody.

In one embodiment, niclosamide, a salicylamide targets the Wnt/β-cateninpathway prepared as a PEGylated lipid nanoemulsion conjugated withcarboplatin, monoclonal antibody, metformin, buformin or phenformincoated pluralities of nanoparticles with cell-penetrating peptides(CPPs), nanoshells, or nanocages administered systemicallyintravenously, intra-arterially locally, intra-arterially locally, inthe body cavity, such as in the bladder, or in the cerebrospinal fluidto attach to the tumor receptors and heated with either an alternatingmagnetic field, focused, or microwaves to heat up the tumors and releasethe medication under control of the temperature, imaged with athermoacoustic, ultrasound imaging system for control of thetemperature, a processor connected to the thermal delivery and thermalimaging unit to release the medication at about 41-43 degrees C. whenthe lipid melts targeting multiple cancers, such as leukemia,glioblastoma or glioma, or ovarian cancer, drug-resistant ovariancancer, ovarian tumor-initiating cells (OTIC) or breast cancer, gastriccancer, colon cancer, or melanomas, etc. and the increased tumorbiomarkers in the circulation after the thermotherapy has an importantdiagnostic (i.e., indicating presence of a tumor) and therapeutic valueas biomarkers for vaccine production with or without VLP attached toantibody coated nanoparticles and administering them with, VLP,checkpoint inhibitors, such as PD-1, Jagged 1 inhibitor 15D11, etc. andRock inhibitors, such as Fasudil, or Wnt inhibitors, such asniclosamide, with cell-penetrating peptides (CPPs) for the managementthe future recurrences in the patient and eliminating the need for CAR-Tadministration to the patient.

In one embodiment, niclosamide, a salicylamide targets the Wnt/β-cateninpathway prepared as PEGylated lipid nanoemulsion conjugated withcarboplatin, or other antineoplastic medications, or temozolomide,monoclonal antibody coated pluralities of nanoparticles withcell-penetrating peptides (CPPs), nanoshells, or nanocages administeredsystemically intravenously, intra-arterially, locally, in the bodycavity, such as in the bladder, mouth, nasal cavity, intraperitoneally,or in the cerebrospinal fluid to attach to the tumor receptors andheated with either an alternating magnetic field, focused ultrasound, ormicrowaves to heat up the tumors and release the medication undercontrol of the temperature, imaged with a thermoacoustic ultrasoundimaging system for control of the temperature with a processor connectedto the thermal delivery and thermal imaging unit to release themedication at about 41-43 degrees C. when the lipid melts targetingmultiple cancers such as leukemia, glioblastoma or glioma, or ovariancancer, drug-resistant ovarian cancer, ovarian tumor-initiating cells(OTIC) or breast cancer, gastric cancer, colon cancer, or melanomas,etc. or inhibit intracellular WNT/CTNNB1-, NOTCH-, mTOR-, and NF-κBsignaling cascades and the increased tumor biomarkers in the circulationafter the thermotherapy has an diagnostic (i.e., indicating presence ofa tumor) and therapeutic value as biomarkers for vaccine production withor without VLP attached to antibody coated nanoparticles, andadministering them with checkpoint inhibitors, such as PD-1, Jagged 1inhibitor 15D11, etc., and Rock inhibitors, such as Fasudil, and lowmolecular weight heparin or IL-6 inhibitors, or Wnt inhibitors, such asniclosamide, with cell-penetrating peptides (CPPs) for the managementthe future recurrences in the patient and eliminating the need for CAR-Tadministration to the patient.

In one embodiment, niclosamide is given orally instead of intravenouslyat the dose of 1-2 grams or less for one day and repeated monthly asneeded simultaneously with thermoimmunotherapy to achieve the killing ofthe tumors such as glioblastoma, ovarian cancer, breast cancer, ormedulloblastoma, and the therapy is repeated as needed.

In one embodiment, in the post-operative period when tumor recurrencesmight occur one can still activate the genetically modified CAR-T cellspresent in the bone marrow of the patient who have been treatedpreviously with CAR-T cells by stimulating their proliferate to attackthe tumor recurrences by using the vaccine prepared from cold storedbiomarkers with or without VLP or oncolytic viruses of the patient'sblood obtained after the initial thermotherapy, and administering themwith checkpoint inhibitors, such as PD-1, Jagged 1 inhibitor 15D11,etc., and Rock inhibitors, such as Fasudil, or Wnt inhibitors, such asniclosamide, conjugated with thermosensitive polymer/quenchedfluorescein or other indicators/medication and the antibody coatedpluralities of nanoparticles, and administering them with checkpointinhibitors, such as PD-1, Jagged 1 inhibitor 15D11, etc., and Rockinhibitors, such as Fasudil, or and low molecular weight heparin or IL-6inhibitors or Wnt inhibitors, such as niclosamide, with cell-penetratingpeptides (CPPs) to achieve release of the vaccine/dye by controlledthermotherapy at the temperature 41-43 degrees C. to damage the tumorcells and stimulating the genetically modified CAR-T cells in the bonemarrow. The vaccination may be repeated in weekly or monthly intervals,as needed, until no sign of the tumor metastasis is found demonstratedby disappearance of the blood biomarkers.

In one embodiment, a known enzyme that dissolves the tumor cells'membrane and mitochondrial cell membrane (i.e., granzyme) is conjugatedwith a thermosensitive polymer antibody/medication coated nanoparticlesand ACPP, gold, silica, magnetic, paramagnetic, and nonmagneticnanoparticles, nanocages or nanoshells, with or without macrolides, suchas cyclosporine A, mycophenolic acid, tacrolimus or ascomycin, havingthe temperature markers quenched fluorescein or other indicators andPFCL, bubble liposomes containing air pockets or nanoemulsions of PFCcarrying fluorescein gene intravenously, intra-arterially locally, or inthe cerebrospinal fluid to attach to the tumor receptors and heated witheither an alternating magnetic field, focused ultrasound, or microwaves,radiofrequency to heat up the nanoparticle/tumors complex and releasethe medication under control of the temperature, and imaged with athermoacoustic imaging system for control of the temperature and aprocessor at 41-43 degrees C. to release the medication/dye and kill thetumor simultaneously by thermal energy in treatment of glioblastoma orglioma, or ovarian cancer or breast cancer, gastric cancer, coloncancer, or melanomas, Mantle cell lymphoma preferentially killing thecancer stem cells, etc. and the increased tumor biomarkers in thecirculation after the thermotherapy has an important diagnostic (i.e.,indicating presence of a tumor) and therapeutic value as biomarkers forvaccine production with or without VLP attached to antibody/medicationcoated nanoparticles and administering them with checkpoint inhibitorssuch as PD-1, Jagged 1 inhibitor 15D11, etc. and Rock inhibitor, Fasudilor Wnt inhibitors, such as niclosamide, with cell-penetrating peptides(CPPs) for the management of the future recurrences in the patient andeliminating the need for CAR-T administration to the patient.

In one embodiment, a known enzyme that dissolves the tumor cells'membrane and mitochondrial cell membrane (e.g., granzyme,metalloproteinase (MMP), chymotrypsin, as pepsin, alpha chymotrypsin, ortrypsin) with or without macrolides, such as cyclosporine A,mycophenolic acid, tacrolimus or ascomycin, is conjugated with athermosensitive polymer antibody/medication coated nanoparticles andACPP, gold, silica, magnetic, paramagnetic, and nonmagneticnanoparticles, nanocages or nanoshells having the temperature markersquenched fluorescein or other indicators and PFCL, or bubble liposomescontaining air pockets or nanoemulsions of PFC carrying fluorescein,gene and intravenously, intra-arterially locally, or in thecerebrospinal fluid to attach to the tumor receptors and heated witheither an alternating magnetic field, focused ultrasound, microwaves, orradiofrequency to heat up the nanoparticle/tumors complex and releasethe medication under control of the temperature, and imaged with athermoacoustic or ultrasound imaging system for control of thetemperature and a processor to release the medication/dye at 41-43degrees C. and kill the tumor simultaneously by thermal energy intreatment of glioblastoma or glioma, or ovarian cancer or breast cancer,gastric cancer, colon cancer, or melanomas, Mantle cell lymphomapreferentially killing the cancer stem cells, etc. and the increasedtumor biomarkers in the circulation after the thermotherapy has animportant diagnostic (i.e., indicating presence of a tumor) andtherapeutic value as biomarkers for vaccine production with or withoutVLP attached to antibody/medication coated nanoparticles andadministering them with checkpoint inhibitors, such as PD-1, Jagged 1inhibitor 15D11, etc. and Rock inhibitors, such as Fasudil, or Wntinhibitors, such as niclosamide, with cell-penetrating peptides (CPPs)for the management of the future recurrences in the patient andeliminating the need for CAR-T administration to the patient.

In one embodiment, a known enzyme that dissolves the tumor cells'membrane and mitochondrial cell membrane (e.g., granzyme,metalloproteinase (MMP), chymotrypsin, as pepsin, alpha chymotrypsin, ortrypsin) is conjugated with a thermosensitive polymerantibody/medication coated nanoparticles and ACPP, VLP, and checkpointinhibitors, but Rock inhibitors, such as Fasudil, etc. given orally orlocally or with gold, silica, magnetic, paramagnetic, and nonmagneticnanoparticles, nanocages or nanoshells having the temperature markersquenched fluorescein or other indicators, such as bubble liposomescontaining air pockets or nanoemulsions of PFC carrying fluorescein geneand drug, with or without macrolides such as cyclosporine A,mycophenolic acid, tacrolimus or ascomycin, intravenously,intra-arterially locally, or in the cerebrospinal fluid to attach to thetumor receptors and heated with either an alternating magnetic field,ultrasound, microwaves, or radiofrequency to heat up thenanoparticle/tumors complex and release the medication under control ofthe temperature, and imaged with a thermoacoustic imaging system forcontrol of the temperature and a processor to release the medication/dyeat 41-43 degrees C. and kill the tumor simultaneously by thermal energyin treatment of glioblastoma or glioma, or ovarian cancer or breastcancer, gastric cancer, colon cancer, or melanomas, Mantle cell lymphomapreferentially killing the cancer stem cells, etc. and the increasedtumor biomarkers in the circulation after the thermotherapy has animportant diagnostic (i.e., indicating presence of a tumor) andtherapeutic value as biomarkers for vaccine production with or withoutVLP attached to antibody coated nanoparticles and administering themwith checkpoint inhibitors such as PD-1, Jagged 1 inhibitor 15D11, etc.and Rock inhibitors, such as Fasudil or Wnt inhibitors, such asniclosamide, with cell-penetrating peptides (CPPs) for the management ofthe future recurrences in the patient and eliminating the need for CAR-Tadministration to the patient.

In one embodiment, the pluralities of antibody/medication coatednanoparticles including piezoelectric nanoparticles carry with theirthermosensitive polymeric coating one or multiple antineoplasticmedications, checkpoint inhibitors, Perforin, cytolysins, complementcomponent 9(C9) granzyme, VLP etc. are used for treatment of cancerusing a combination of focused ultrasound, alternating magnetic field,or electromagnetic radiation, non-invasively at a temperature 41-43degrees C. under the control of a processor controlling the thermalenergy, imaged with a thermoacoustic imaging system for control of thetemperature, along with low-intensity (1-3 V/cm), intermediate frequency(100-300 kHz), electric fields achieving thermotherapy, electroacousticimaging and dielectric effect on the cellular components of the tumors,such as brain tumors, lung cancer, ovarian cancer, breast cancer,prostate cancer, and other cancers, which can be repeated numerous timespost therapy to eliminate the cancer with simultaneous aspiration of alysed tumor mass through a 20-30 gauge needle to reduce the toxic burdenon the body while the increased tumor biomarkers in the circulationafter the thermotherapy has an important diagnostic (i.e., indicatingpresence of a tumor) and therapeutic value as biomarkers for vaccineproduction with or without VLP attached to antibody coatednanoparticles, and administering them with checkpoint inhibitors such asPD-1, Jagged 1 inhibitor 15D11, etc. and Rock inhibitor, such asFasudil, and low molecular weight heparin or IL6 inhibitors or Wntinhibitors, such as niclosamide, with cell-penetrating peptides (CPPs)for the management of the future recurrences in the patient andeliminating the need for CAR-T administration to the patient, whiledoing simultaneously plasmapheresis, kidney dialysis, dielectrophoresisof the blood clears tumor toxins, dead killer cells, etc. from the bloodand prevents a cytotoxic toxic storm and systemic medication withtocilizumab, a monoclonal antibody that targets interleukin-6 andornithine phenylacetate an ammonia scavenger, or ARF6 inhibition fortreatment of hepatic encephalopathy, a neuropsychiatric syndromeassociated with hyperammonemia.

In one embodiment, a known enzyme that dissolves the wall of thebacteria, viruses, fungi, parasites, such as MRSA, granzyme,metalloproteinase (MMP), chymotrypsin, as pepsin, alpha chymotrypsin,trypsin conjugated with a thermosensitive polymer antibody/medicationcoated nanoparticles of iron, iron oxide, gold, silica, magnetic,paramagnetic, and nonmagnetic nanoparticles, nanocages or nanoshellshaving the temperature markers quenched fluorescein or other indicatorsor with Liposomes having temperature triggered opening of a leucinezipper peptide inserted in the membrane of a dye and medication carryinga liposome opens a channel through which the drug is released at thetemperature or 41-43 degrees C. or opens drug-permeable pores createdbubble formation from the decomposition of encapsulated ammoniumbicarbonate injected intravenously, intra-arterially, locally, or in thecerebrospinal fluid to attach to the bacterial surface antigens andheated with either an alternating magnetic field, ultrasound,microwaves, or radiofrequency to heat up the nanoparticle/bacteria,viruses complex and release the medication under control of thetemperature, and imaged with a thermoacoustic ultrasound imaging systemfor control of the temperature and a processor to release themedication/dye at 41-43 degrees C. to damage the wall of bacteria,viruses, etc. kill them simultaneously by thermal energy treatment inthe patient, while injecting simultaneously locally, intravenously rockinhibitors, GSK inhibitors, integrin inhibitors and low molecular weightheparin or IL-6 inhibitors to reduce inflammatory process and doingsimultaneously plasmaphoresis, kidney dialysis, dielectrophoresis of theblood clears tumor toxins, dead killer cells, etc. from the blood andprevents a toxic storm and prevents damage to the kidney, liver, heart,etc. and systemic medication with tocilizumab, a monoclonal antibodythat targets interleukin-6 and ornithine phenylacetate an ammoniascavenger, for treatment of hepatic encephalopathy, a neuropsychiatricsyndrome associated with hyperammonemia or ARF6 inhibition.

In one embodiment, thermal and electrical energy is used to influencethe permeability of the tumor cell membrane so as to utilize the effector thermotherapy with pluralities of nanoparticles or liposomes havingtemperature triggered opening of a leucine zipper peptide inserted inthe membrane of a dye and medication carrying liposome opens a channelthrough which the drug is released at the temperature or 41-43 degreesC. or opens drug-permeable pores created bubble formation from thedecomposition of encapsulated ammonium bicarbonate injected in the bodyto enhance the effect of chemotherapy or immune therapy, gene therapy,gene modification using antibody coated nanoparticles conjugated withCRISPR-cas9 in malignant cells, such as brain and spinal cord tumors,breast cancer, lung cancer, prostate and ovarian cancer and melanoma,glioblastoma, retinoblastoma, meduloblastoma, gastrointestinal andgenitourinary tumor of sarcoma, or benign expanding tumor, such asmemningioma, uterus fibroma, etc., based on the antibody coatedconjugated nanoparticles with CRISPR/Cas9 system, in which a DNA-cuttingCas9 enzyme targets the checkpoint inhibitor regions removing checkpointinhibitors, but Rock inhibitors, such as Fasudil, etc. given orally orlocally, or systemically, genes of the tumor cells and replace them withsuicide genes defined as genes whose products cause cell death, e.g.,thymidine kinase (HS-TK), cytosine deaminase, etc., pro-apoptatic genes,etc. through antibody coated nanoparticles conjugated with CRISPR cas9mediated Homology-Independent Targeted Integration (HITI) or HomologyDirected Repair (HDR) further enhancing an immune response which is notbeing blocked by the checkpoint inhibitors of the tumor cells. Antibodycoated nanoparticles are used having pyroelectric or piezoelectriccharacteristics, and the antibody coated nanoparticle tube complex isimaged with an electroacoustic computed tomography imaging system.

In one embodiment, the antibody/medication coated pluralities of gold,piezoelectric, iron oxide, nanocage, nanotube, nanoshell, magnetic,paramagnetic, pyroelectric and/or piezoelectric nanoparticles are coatedwith thermosensitive polymers, or with liposomes having temperaturetriggered opening of a leucine zipper peptide inserted in the membraneof a dye and medication carrying liposome opens a channel through whichthe drug is released at the temperature or 41-43 degrees C. or opensdrug-permeable pores created bubble formation from the decomposition ofencapsulated ammonium bicarbonate or polymers, such as chitosan,quenched with fluorescein or other indicators conjugated withantineoplastic medication, gene, CRISPR-cas9 conjugated with cellpenetrating agents or activatable cell penetrating agents injected inthe patient's circulation, lymphatic vessels, inside a body cavity,cerebrospinal fluid, etc. is released that can be revealed when thenanoparticles are exposed to electromagnetic radiation, combined focusedultrasound in a thermal or non-thermal mode or non-focused ultrasound,or electrical current generated by a battery where low electricalcurrent from a battery passes from one side of the skin (i.e., theanode) through the body and a lesion or tumor to the cathode electrodepositioned on the opposite side of the skin on the body to raise thetemperature of the pluralities of the piezoelectric or pyroelectricnanoparticles that are injected inside the body to be attached to thesurface antigens of the normal cells or of tumor cells and createnanoparticle/tumor cell complexes to 41-43 degrees C., and when exposedto pulses of electrical current with an adjustable signal frequency andvoltage, an acoustic response is produced by electrical stimulation ofpiezoelectric nanoparticle inside the body that is called anelectroacoustic sound or signal which can be recorded with a transducer,or microphone, converted to an electrical signal and forwarded to aprocessor to be converted to a 1D, 2D, or 3D image as an electroacousticcomputed tomogram while the electrical pulse generated in thepiezoelectric nanoparticles drives the medication or gene in the tumorcells locally to damage the tumor cells by multiple modes of the therapyapplied non-invasively based on the antibody coated nanoparticlesconjugated with the CRISPR/Cas9 system, in which a DNA-cutting Cas9enzyme targets the checkpoint inhibitor regions of the tumor cellsremoving checkpoint inhibitors genes and replacing them with suicidegenes, defined as genes whose products cause cell death, e.g., thymidinekinase (HS-TK), cytosine deaminase, etc., pro-apoptic genes, etc.through the antibody coated nanoparticles conjugated with CRISPR cas9mediated Homology-Independent Targeted Integration (HITI) or HomologyDirected Repair (HDR) further enhancing an immune response which is notbeing blocked by the checkpoint inhibitors of the tumor cells incombination with, Rock inhibitors, such as Fasudil, etc. given orally orlocally or systemically to reduce inflammatory cells and inhibitproduction of TGF-β and fibrous tissue.

In one embodiment, during the thermal delivery to theantibody/medication coated nanoparticles, nanoshells, or nanocages,magnetic, non-magnetic, MNP, paramagnetic, organic piezoelectric,pyroelectric, fluorine, dendrimers, quartz or combinations thereof, orwith liposomes having temperature triggered opening of a leucine zipperpeptide inserted in the membrane of a dye and medication carryingliposome opens a channel through which the drug is released at thetemperature or 41-43 degrees C. or opens drug-permeable pores createdbubble formation from the decomposition of encapsulated ammoniumbicarbonate, conjugated with a thermosensitive polymer, such aschitosan, or a medication, gene, etc., or bubble liposomes containingair pockets or nanoemulsions of PFC carrying fluorescein gene, and aftertheir injection in the body, one can measure two distinct temperaturesinside the body regardless of their location using either anelectromagnetic, focused ultrasound, or alternating magnetic field,while releasing the medications, imaged with a thermoacoustic imagingsystem for control of the temperature at a temperature of 41-43 degreesC., this can achieve a theranostic or diagnostic and therapeutic effectwhen the nanoparticles with activatable cell-penetrating peptides(ACPPs) carry a thermosensitive polymer, such as chitosan, and a dye oranother dye or indicator etc., such as quenched fluorescein, or otherindicators that can be only detected when it is released in the bloodindicating the melting temperature of the thermosensitive coating orthat 41-43 degrees C. is reached and the nanoparticle release of themedication conjugated with the chitosan by detection of unquenchedfluorescein or another dye or indicator present in the bloodcontinuously passing through a flexible silicone tube, nail bed, or anyother part of the body, radiated by UV radiation or light of anotherwavelength to fluoresce, or other dyes, such as indocyanin green atinfrared wavelengths using the nail bed or any other visible vesselssuch as conjunctival vessels, etc. for illumination and recording thefluorescence wavelength through a filter that eliminates the radiatingfrom the fluorescent wave length indicating release of dye, medication,gene etc. further heating as needed at the areas of the body dependingon the thermal energy delivery source or combinations with LIFU modeultrasound, laser, microwave, radiofrequency (RF), alternating magneticfield, or any other source of the energy from outside the body or insidethe body, and imaged with a thermoacoustic or photoacoustic imagingsystem for control of the temperature.

In one embodiment, thermal and electrical energy is used to influencethe permeability of the tumor cell membrane so as to utilize the effector thermotherapy to enhance the effect of chemotherapy or immunetherapy, gene therapy, gene modification using CRISPR-cas9 in malignantcells, such as brain and spinal cord tumors, breast cancer, lung cancer,prostate and ovarian cancer and melanoma, glioblastoma, retinoblastoma,meduloblastoma, gastrointestinal and genitourinary tumor of sarcoma, orbenign expanding tumors, such as meningioma, uterus fibroma, etc., basedon the antibody coated nanoparticles conjugated with CRISPR/Cas9 system,in which a DNA-cutting Cas9 enzyme or combination withantibody/medication coated nanoparticles or with liposomes injected inthe body having temperature triggered opening of a leucine zipperpeptide inserted in the membrane of a dye and medication carryingliposome opens a channel through which the drug is released at thetemperature or 41-43 degrees C. or opens drug-permeable pores createdbubble formation from the decomposition of encapsulated ammoniumbicarbonate, targets the checkpoint inhibitor regions removingcheckpoint inhibitors genes of the T-lymphocytes and killer cells orCAR-cells to make them non-responsive to the tumor checkpoint inhibitorsof the tumor cells through CRISPR cas9 mediated Homology-IndependentTargeted Integration (HITI) or Homology Directed Repair (HDR) furtherenhancing an immune response which is not being blocked by thecheckpoint inhibitors of the tumor cells. Antibody coated nanoparticlesare used having pyroelectric or piezoelectric characteristics, and thenanoparticle tube complex is imaged with an electroacoustic computedtomography imaging system.

In one embodiment, thermal and electrical energy is used to influencethe permeability of the tumor cell membrane so as to utilize the effector thermotherapy to enhance the effect of chemotherapy or immunetherapy, gene therapy, gene modification using antibody coatednanoparticles having pyroelectric or piezoelectric characteristics, andthe nanoparticle tube complex is imaged with an electroacoustic computedtomography imaging system and conjugated with CRISPR-cas9 mediatedHomology-Independent Targeted Integration (HITI) or Homology DirectedRepair (HDR) further enhancing an immune response which is not beingblocked by the checkpoint inhibitors in malignant cells, such as braintumors and spinal cord tumors, breast cancer, lung cancer, prostate andovarian cancer and melanoma, glioblastoma, retinoblastoma,meduloblastoma, gastrointestinal and genitourinary tumor of sarcoma, orbenign expanding tumors, such as meningioma, uterus fibroma, etc., basedon the antibody coated nanoparticles and liposome with temperaturetriggered opening of a leucine zipper peptide inserted in the membraneof a dye and medication carrying liposome opens a channel through whichthe drug is released at the temperature of 41-43 degrees C. or opensdrug-permeable pores created bubble formation from the decomposition ofencapsulated ammonium bicarbonate, after heating with an energy sourcetargets, or in vitro tissue culture.

In one embodiment, in vitro culture sensitization to tumor antigen andVLP coated nanoparticles and simultaneous modification of T-cells withCRISPR Cas9 with antibody/medication coated nanoparticles or withliposomes injected in the tissue culture having temperature triggeredopening of a leucine zipper peptide inserted in the membrane of a dyeand medication carrying liposome after heating with an energy sourceopens a channel through which the drug is released at the temperature of41-43 degrees C. or opens drug-permeable pores created bubble formationfrom the decomposition of encapsulated ammonium bicarbonate, eliminatingcheckpoint inhibitors of the T-lymphocytes, killer cells, Car-cellsresulting in losing their response to checkpoint inhibitors of a tumorwhile maintaining T-Cell action against the tumor, preventingautoimmunity formation and the attacking normal cells in the body, andusing the same concept for tumor vaccination with or without presence ofa new metastatic disease.

In one embodiment, the systemic administration of antibody coatednanoparticles conjugated with CRISPR Cas9 cells eliminating the body'scheckpoint inhibitor of the immune responsive cells, usingantibody/medication coated nanoparticles or liposomes in treatment ofsevere autoimmune diseases involving the digestive tract, centralnervous system, such as multiple sclerosis and Gillian-barre syndrome orrespiratory diseases, such as asthma and arthritis, giant cell arthritisand arteriosclerosis, etc. in vivo to replace the normal immune cellswith immune cells with the immune responsive cells without checkpointinhibitors that are not recognizing the normal cells or patient's owncells as foreign, but still maintain their response to other antigeniccompounds, such as VLP or oncolytic viruses such as reoviruses, etcconjugated with an antibody to tumors, bacteria, injected systemicallylocally inside the tumor, intravenously or intra-arterially, etc., or intreatment of severe autoimmune diseases involving digestive tract,central nervous system, such as multiple sclerosis and Gillian-barresyndrome or respiratory diseases, such as asthma and arthritis, giantcell arthritis and arteriosclerosis, cardiovascular inflammation,psoriasis, uveitis, etc. eliminating autoimmunity in these diseases.

In one embodiment, in metastatic diseases and potential cytokine storm,one can induce in addition to vaccine therapy an oral immunity bygrowing the patient's tumor cell in sufficient quantity in vitro andradiating them by cobalt beta or exposing them to another source ofthermal or non-thermal radiation to damage the genetic component of thetumor or kill them, while maintaining the antigenicity of the tumor cellproteins, then administer them orally after encapsulating them with Rockinhibitors such as Fasudil, and low molecular weight heparin or IL-6inhibitors or other small molecule Rho kinase inhibitors or GSK 269962inhibitor in addition to oral Wnt inhibitors, such as ivermectin,niclosamide, apicularen and bafilomycin, to reduce inflammatory andTGF-β production after, subcutaneous, intramuscular or intravenous orintraperitoneal vaccination in the patient who have developed anautoimmune response after immune therapy.

In one embodiment, in a patient with metastatic disease where a cytokinestorm can be expected after standard immunotherapy, antibody coatednanoparticles are conjugated with non-toxic dose of Rock inhibitors, andlow molecular weight heparin or IL-6 inhibitors, Wnt inhibitors,combined with macrolide immunosuppressants such as cyclosporine,mycophenolic acid, tacrolimus, ascomycin etc. to suppress an excessiveimmune response and inflammation in the normal tissue by blocking theIL-17 from the memory Th17 cells with the existing severe immuneresponse in the skin, gut, joint, etc.

In one embodiment, in a patient with metastatic disease, the use ofantibody coated pluralities of nanoparticles can be conjugated with Rockinhibitors, and low molecular weight heparin or IL-6 inhibitors, Wntinhibitors and GSK 269962 inhibitors along with antibody coatednanoparticles with cyclosporine A, or mycophenolic acid or tacrolimusetc. to significantly damage the tumor cells and their metastatic lesionwith or without thermotherapy.

In one embodiment, the treatment of a tumor with a metastatic lesion isdone with simultaneous administration of pluralities of the antibodycoated nanoparticles conjugated with various medications such as Wntinhibitors, GSK inhibitors, or Rock inhibitors with macrolide antibodiessuch as cyclosporine A, Ascomycin, mycophenolic acid or tacrolimus, antiVEGFs such as Avastin, etc. and simultaneous administration ofcheckpoint inhibitors, such as PD-1, PD-L1, CTLA-4, Jagged 1 inhibitor15D11, etc. and immune stimulators, such as VLP, IL-2, IL-4 TLR7 etc.and simultaneous subcutaneous or intra-tumoral or intravenousvaccination with antibody coated nanoparticles conjugated with VLP, orother modified Reoviruses, or granzyme and IL2, IFN-γ, TLR 4,7 whereafter the initial therapy the vaccination will continue at weekly, thenmonthly, then every 3 month intervals and evaluated by presence of thecirculating tumor exosome, or circulating tumor cells or presence oftumor cells that have devoured the macrophages or other normal cells(e.g., lymphocytes) thereby becoming hybrid tumor cells diagnosed byobtaining blood from the patients in the postoperative period, thepresence of the patient and tumor genetic material in these cells,incubating the tumors in the culture and thermosensitive nanoparticles,administering laser radiation to kill these cells and obtain new lysatesto produce a new vaccine. In one embodiment, as a routineplasmaphoresis, dielectrophoresis, and kidney dialysis is done to removethe dead normal cells and hybrid cells and exosomes, and toxins, etc.from the blood that can still cleaned and repurposed to the patient.

In one embodiment, the antibody obtained from the patient's body or intissue culture from the hybrid cells are used to coat the nanoparticlesconjugated with CRSPR cas9 and with cytotoxic siRNA to damage the hybridtumor cell. In one embodiment, the nanoparticles carry both the tumorantibody and the hybrid cell antibody to deliver cytotoxic siRNA to bothkinds of the cells and protect the normal cells. In one embodiment, thisprocedure is repeated with simultaneous thermotherapy with an externalsource of energy to damage these cells, and the treatment is repeatedweekly or monthly along with either Rock inhibitors, Wnt inhibitors, orGSK 269962 inhibitor lithium along with antibody coated nanoparticles toinhibit GSK and repeated until the hybrid cells or tumor cells are notfound from the patient's blood samples.

In one embodiment, the combination therapy for the late metastaticdisease may produce an autoimmune response. In this case, the genetherapy is combined with Rock inhibitors, etc. and macrolides to reducepost-treatment immune and inflammatory responses.

In one embodiment, the pluralities of antibody coated nanoparticles areconjugated with Rock inhibitors, Wnt inhibitors, GSK inhibitors,anti-VEGFs, such as Avastin, NSAIDS such as Diclofenac, acetylsalicylicacid, aspirin, Lovastatins to block phosphoinositol activation as aresults of hypoxia inducible factor-1 which induces VEGF production.

In one embodiment, HIF1A induces Myelodysplastic Syndromes which can betreated with antibody coated nanoparticles conjugated with Wntinhibitors, GSK269962, inhibitor and anti-VEGFs.

In some cancer patients, there is a deficiency of cyclin-dependentKinase 12 (CDK12) which is involved in repair of DNA that can bediagnosed by genomic analysis. The deficiency or mismatch of CDK12 hasbeen found in numerous tumors, such as cancer of the prostate, breast,gastrointestinal tract, bladder, uterine, and ovarian cancers thatrespond positively to the administration of poly(adenosine diphosphate[ADN]-ribose) polymerase (PARP) inhibitors or platinum chemotherapiessuch as cisplatin, carboplatin. In one embodiment, pluralities ofantibody coated nanoparticles are conjugated with a polymer carryingRock or Wnt of GSK inhibitors and PARP and or platinum chemotherapy andVLP or oncolytic viruses, anti-VEGFs and checkpoint inhibitors injectedlocally or systemically, intravenously or preferably intra-arterially,into the feeding artery, applying and exposing the nanoparticles to thetumor cells prior to their systemic uptake by the Reticuloendothelialcells (REC) applying optionally external thermotherapy, laser, LIFU,HIFU, alternating magnetic field in non-thermal or thermal frequencieswhile controlling the temperature with a thermal imaging unit and asoftware from 37 degrees C. to 41 degrees C. or more as needed,releasing the medication to kill damaged tumor cell and their exosomesor CTC or their exosomes.

In one embodiment, excessive production of cytokines from the tumor oras a result of therapy change the tumor microenvironment, and as aresult, the cytotoxic T lymphocytes become exhausted and lose theirnormally present anti-tumor action and, in contrast, produce on theirsurface membrane inhibitory makers and angiogenetic factors, and cannotproduce other anti-tumor chemokines, such as IFN-γ or granzyme β thus,encouraging tumor resistance and development of metastatic disease orchemotherapy resistant tumor cells, release of IL-1 activates NF-κBencouraging metastatic disease and limits the use of immunotherapy withT Cell Receptor (TCR) or chimeric antigen receptor (CAR) in thesepatients. In one embodiment, with exhausted cytotoxic lymphocytes, thepatient is treated with antibody coated nanoparticles conjugated withRock inhibitors, such as Fasudil, etc. or Wnt inhibitors or GSK269962inhibitors with or without macrolides and subsequent plasmaphoresis ordielectrophoresis to remove toxins and dead cells from the patient'sbody, and the clean blood can be re-infused to the patient. In oneembodiment, the treatment is systemic, and in another embodiment, thetreatment is done locally, or orally combined with anti-VEGF coatednanoparticles.

In another embodiment, the pluralities of antibody coated nanoparticlesare conjugated with Wnt inhibitors, Rock inhibitors or GSK inhibitors incombination with a MAP/ERK inhibitor such as sorafenib an RAF kinaseinhibitor or Raf inhibitor sB 590885, PLX4720, RAF 265, XL281,ecorafenib, darafenib,vemurafenib or MEK inhibitors such as cobimetinib,Bimimetinib, selumetinib or Trametinib or GSK 112020212 along with ananti-VEGF, such as Avastin, and checkpoint inhibitors, and IL-2, VLP, oroncolytic viruses such as Reovirus, etc. in a thermosensitive polymericcoating to release the medication after thermotherapy at temperatures of39 to 43 degrees C. or more to damage the tumor cells but control theinflammatory process and prevent a cytokine storm formation orautoimmune response.

Although the antibodies found on the regular tumor cell can be grown onthe cancer stem cells (CSC), it may not be easy to target these cells.In one embodiment, the antibody coated nanoparticles are conjugated witha slow release polymer, such as polylactic or glycolic acid or poroussilicon that can stay around the tumor cells or CSC and theirvasculature to damage the genetic components of the cancer stem cellsand their vascular supply, while carrying VLP or oncolytic viruses,checkpoint inhibitors and Rock inhibitors, Rho-kinases GSK 269962,potent and selective Rock inhibitor GSK 429286, Selective Rho-kinase(ROCK) inhibitor H1152 dihydrochloride, Selective Rho-kinase (ROCK)inhibitor Glycyl H 1152 dihydrochloride, Selective Rho-kinase (ROCK)inhibitor; more selective analogue of H1152, Cell-permeable, selectiveRho-kinase inhibitor OXA 06 dihydrochloride, potent ROCK inhibitorPKI1447 dihydrochloride, potent and selective ROCK inhibitor; antitumorSB 772077B, potent Rho-kinase inhibitor; vasodilator SR 3677dihydrochloride, potent, selective Rho-kinase (ROCK) inhibitorTC-57001,potent and highly selective ROCK inhibitor; orally active Y-27632dihydrochloride, and low molecular weight heparin or IL-6 inhibitors orWnt inhibitors, such as FH535, IWP-2, PNU-74654, IWR-1endo, IWR-exo,Demethoxycurcumin, CCT036477, KY02111, WAY-316606, SFRP, IWP, LGK974,C59, Ant1.4Br/Ant 1.4Cl, ivermectin, niclosamide, apicularen andbafilomycin, XAV939, XAV939, G007-LK and G244-LM, NSC668036, SB-216763,gemtuzumab, or GSK inhibitors GSK 429286, and Avastin an anti-VEGF toinduce an immune stimulation locally but control the inflammation andfurther damage or kill the tumor by stimulation of an immune responsewhile the existence of the radioactive coating, such as functionalizedradioactive gold or combination of gold and ferric oxide nanoparticleswith activatable cell-penetrating peptides (ACPPs) or alpha or a betaradiator, of the nanoparticles helps with the continuous radiation evenif they are picked up by the cytotoxic lymphocytes, macrophages, orkiller cells, preventing them from becoming either a victim of the tumorcell or to be converted to a hybrid tumor cell, while continue locallyradiating the CSC or tumor cells from inside them and killing the tumorcell along with the patient's immune cells by the effect of thelocalized radiation and kill them so as to be removed by either cellularimmune response or plasmaphoresis or dielectrophoresis and remove thereleased toxins from the circulation.

In one embodiment, pluralities of radioactive functionalizednanoparticles are used to treat hybrid tumor cells in conjunction withan immunotherapy treatment method that administers nanoparticlescontaining a radioactive material to the body of a cancer patient. Inthis embodiment, the nanoparticles containing the radioactive materialare conjugated with cell penetrating peptides (CPPs) or activatable cellpenetrating peptides (ACPPs) for penetration and uptake into the hybridtumor cells. In this embodiment, after the uptake of the nanoparticlescontaining the radioactive material in the hybrid tumor cells, theradioactive material is disposed inside the hybrid tumor cells. Onceinside the hybrid tumor cells, the radioactive material of thenanoparticles damages the genetic component of the hybrid tumor cells,thereby preventing the proliferation of the hybrid tumor cells andenabling the hybrid tumor cells to be destroyed.

In one embodiment, viruses are causes of the development of the cancer,such as Merkel-cell carcinoma, papilloma virus, Herpes virus,Creutzfeldt-Jakob disease, Epstein-Barr virus, that can produce bothskin cancer or brain cancer, or may lead to neurodegenerative processes,such as Alzheimer's disease, similarly the prions can cause inflammationin the brain, the inflammatory process caused by the viruses, or prionsactivates p38 MAPK synaptotoxic signaling pathway and causes neuronaldamage, Rock inhibitors control the inflammatory process and preventcell death particularly contributing to neuronal regeneration andbranching of the axons, similarly GSK-3 beta inhibition by Lithiumcauses spreading the cerebellar neurons and has implication in treatmentof Alzheimer's disease, or Parkinson disease. In one embodiment, theRock inhibitor or GSK inhibitors conjugated with polymeric nanoparticlesuch as polylactic or glycolic acid can be administered intranasally totravel to the brain via the olfactory nerve.

Any of the features or attributes of the above described embodiments andvariations can be used in combination with any of the other features andattributes of the above described embodiments and variations as desired.Also, as it is used throughout this disclosure, the conjunction “and/or”means that at least one item in a recited group is included, but up toall items in the recited group can be included (e.g., “nanoparticles,liposomes, and/or micelles” can mean (i) nanoparticles, liposomes, ormicelles; or (ii) nanoparticles, liposomes, and micelles).

The embodiments shown and described in the specification are onlyspecific embodiments of the inventor who is skilled in the art and arenot limiting in any way. Therefore, various changes, modifications, oralterations to those embodiments may be made without departing from thespirit of the invention in the scope of the following claims. Thereferences cited are expressly incorporated by reference herein in theirentirety.

What is claimed is:
 1. A cancer treatment method using controlledlocalized thermotherapy, the method comprising the steps of:administering a plurality of nanoparticles, liposomes, and/or micellesto a patient in need thereof so as to target a tumor in the patient, theadministered nanoparticles, liposomes, and/or micelles being conjugatedwith antitumor antibodies or aptamers, and the administerednanoparticles, liposomes, and/or micelles containing a medication and/orgene, the plurality of nanoparticles being coated with a thermosensitivepolymer, at least some of the antibody or aptamer-conjugatednanoparticles, liposomes, and/or micelles attaching to surface antigensof tumor cells of the tumor so as to form a tumorcell/nanoparticle/liposome/micelle complex; heating the plurality ofnanoparticles, liposomes, and/or micelles with an energy source to atemperature of about 41° C. to about 43° C. so as to damage one or moretumor cell membranes at a treatment site of the tumor and melt thethermo sensitive polymer coating of the nanoparticles and/or break apartthe liposomes and/or micelles, thereby releasing the medication and/orgene at the treatment site of the patient; measuring the temperature atthe treatment site of the patient with photoacoustic imaging using alaser, or with an ultrasound transducer that measures a variation in theattenuation coefficient, a change in backscattered energy of aultrasonic signal, a backscattered radio-frequency echo-shift due to achange in the speed of sound and thermal expansion of the tissue, and/ora change in the amplitudes of the acoustic harmonics; and controllingthe energy source based upon the measured temperature so as to maintainthe temperature at the treatment site within the range of about 41° C.to about 43° C.
 2. The cancer treatment method according to claim 1,wherein a plurality of liposomes are administered to the patient, atleast some of the plurality of the liposomes being filled with themedication and/or gene.
 3. The cancer treatment method according toclaim 1, wherein a plurality of nanoparticles are administered to thepatient, the plurality of nanoparticles being selected from a groupconsisting of iron oxide gold nanoparticles, graphene oxidenanoparticles, gold nanoparticles, silicon nanoparticles, carbonnanoparticles, magnetic nanoparticles, gold nanorods, gold nanoshells,gold nanocages, iron oxide nanotubes, gold nanotubes, carbon nanotubes,quartz, and combinations thereof.
 4. The cancer treatment methodaccording to claim 3, wherein the nanoparticles are further conjugatedwith cell penetrating peptides (CPPs) or activatable cell-penetratingpeptides (ACPPs) so to enhance cell penetration into the cells of thetumor.
 5. The cancer treatment method according to claim 1, wherein thestep of heating the plurality of nanoparticles, liposomes, and/ormicelles releases tumor antigens in the circulation of the patient as aresult of thermally damaging the tumor cells; and wherein the methodfurther comprising the steps of: obtaining from the blood of thepatient, the tumor antigens to build a vaccine against tumor specificantigens, the vaccine combined with antibody or aptamer-conjugatednanoparticles, liposomes, and/or micelles that are conjugated withcheckpoint inhibitors, Rock inhibitors, IL-6 inhibitors, and/or Wntinhibitors; administering the vaccine inside the tumor,intra-arterially, intravenously or subcutaneously with the antibody oraptamer-conjugated nanoparticles, liposomes, and/or micelles conjugatedwith viral-like particles (VLP) and/or oncolytic viruses whilesimultaneously releasing conjugated checkpoint inhibitors, Rockinhibitors, IL-6 inhibitors, and/or Wnt inhibitors from the antibody oraptamer-conjugated nanoparticles, liposomes, and/or micelles to preventnew or old tumor cells, metastatic cells, and/or tumor exosomes frombeing disguised from the T-lymphocytes or the patient's natural killer(NK) cells, thereby providing a vaccine for treatment of potentialrecurrences of the same tumor to the patient and enhancing the immuneresponse at the specific location of one or more metastatic lesions,circulating tumor cells, or sessile tumor cells; and heating theantibody or aptamer-conjugated nanoparticles, liposomes, and/or micellesand the viral-like particles (VLP) and/or oncolytic viruses of thevaccine so as to kill the VLP and/or oncolytic viruses while leaving theantigenic foreign proteins of the VLP and/or oncolytic viruses at thetumor site to enhance an immune response of the patient.
 6. The cancertreatment method according to claim 1, wherein the medication isconjugated with the nanoparticles and/or disposed inside the liposomes,the medication being selected from the group consisting of Wntinhibitors, Rock inhibitors, IL-6 inhibitors, low molecular weightheparin, metformin, buformin, syrosingopine, phenformin, anti-vascularendothelial growth factors (anti-VEGFs), checkpoint inhibitors,macrolides, glycogen synthase kinase (GSK) inhibitors, and combinationsthereof.
 7. The cancer treatment method according to claim 1, wherein aplurality of liposomes are administered to the patient, at least some ofthe plurality of the liposomes being filled with nanoparticles coatedwith a slow release polymer, the slow release polymer being selectedfrom the group consisting of polycaprolactone, polylactic acid, andpolyglycolic acid, and the nanoparticles disposed in the liposomescontaining doxorubicin and porphyrin as a cell penetrating agent, wherethe porphyrin attaches to the cell membrane proteins of the tumor cellswhen released from the liposomes after being heating to a temperature ofabout 40° C. to about 43° C., and the doxorubicin entering the tumorcells with ease so as to damage the tumor cells.
 8. The cancer treatmentmethod according to claim 1, wherein the energy source for heating theantibody or aptamer-conjugated nanoparticles, liposomes, and/or micellesis selected from the group consisting of ultrasound, laser, analternating magnetic field, microwave radiation, and radiofrequency (RF)energy.
 9. The cancer treatment method according to claim 1, wherein theenergy source for heating the antibody or aptamer-conjugatednanoparticles, liposomes, and/or micelles is controlled by aproportional-integral-derivative (PID) controller for maintaining thetemperature at the treatment site within the range of about 41° C. toabout 43° C. temperature.
 10. The cancer treatment method according toclaim 1, wherein the antibody or aptamer-conjugated nanoparticles,liposomes, and/or micelles are administered inside the tumor,intra-arterially near the tumor, and/or intravenously.
 11. The cancertreatment method according to claim 1, wherein the medication isconjugated with the nanoparticles and/or disposed inside the liposomes,the medication comprising low molecular weight heparin for reducing aninflammatory response in the tissue, sodium bicarbonate to reduce pH ofthe inflamed tissue, and IL-6 inhibitors to dampen a postoperativecytokine inflammatory response.
 12. A cancer treatment method comprisingadministering to a patient having an early stage tumor a combination ofthermotherapy and immunotherapy, where administering a plurality ofnanoparticles, liposomes, and/or micelles to a patient in need thereofso as to target a tumor in the patient, the administered nanoparticles,liposomes, and/or micelles being conjugated with antitumor antibodies oraptamers, and the administered nanoparticles, liposomes, and/or micellescontaining a medication and/or gene, the plurality of nanoparticlesbeing coated with a thermosensitive polymer, at least some of theantibody or aptamer-conjugated nanoparticles, liposomes, and/or micellesattaching to surface antigens of tumor cells of the tumor so as to forma tumor cell/nanoparticle/liposome/micelle complex; heating theplurality of nanoparticles, liposomes, and/or micelles with an energysource to a temperature of about 41° C. to about 43° C. so as to damageone or more tumor cell membranes at a treatment site of the tumor andmelt the thermo sensitive polymer coating of the nanoparticles and/orbreak apart the liposomes and/or micelles, thereby releasing themedication and/or gene at the treatment site of the patient; andimmunotherapy comprises systemically administering the patient's naturalkiller (NK) cells/dendritic cells pre-sensitized in vitro to the tumor.13. The cancer treatment method according to claim 12, wherein themethod further comprises the step of: removing cytokines after theimmunotherapy by electrophoresis, plasmapheresis, or plasma exchange soto prevent a cytokine storm.
 14. The cancer treatment method accordingto claim 12, wherein the antibody or aptamer-conjugated nanoparticles,liposomes, and/or micelles are further conjugated with an inhibitorygene(s) and a CRISPR/cas9 complex to stimulate or modify tumor genes atthe site of the tumor upon release from the thermosensitive polymercoating of the nanoparticles, or upon release from the liposomes ormicelles, at a temperature of about 41° C. to about 43° C.
 15. Thecancer treatment method according to claim 14, where gene modificationis done using CRISPR/cas9 mediated homology-independent targetedintegration (HITI) or homology directed repair (HDR).